Protein material

ABSTRACT

A method of treating a proteinaceous material having a first concentration of β-conglycinin, the method including combining the proteinaceous material with an enzyme to form a reaction mixture, the reaction mixture initially having a pH of at least about 7.0 standard pH units, allowing the enzyme to hydrolyze β-conglycinin present in the reaction mixture to form a proteinaceous intermediate, and inactivating the enzyme present in the reaction mixture after a reaction period to form a proteinaceous product, the proteinaceous product having a second concentration of β-conglycinin, the second concentration of β-conglycinin being at least 99 percent less than the first concentration of β-conglycinin.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of priority from U.S. patentapplication Ser. No. 60/199,758 that was filed on Apr. 26, 2000 and alsoclaims the benefit of priority from PCT International Application Ser.No. PCT/US01/13372 that was filed on Apr. 26, 2001, since thisapplication is a National Phase application into the U.S. filed under 35U.S.C. § 371 from PCT International Application Ser. No. PCT/US01/13372that was filed on Apr. 26, 2001 and since PCT International ApplicationSer. No. PCT/US01/13372 claims the benefit of priority from U.S.Provisional Application Ser. No. 60/199,758 that was filed on Apr. 26,2000.

BACKGROUND OF THE INVENTION

The present invention generally relates to a method of reducing theantigenicity of vegetable proteins, while also improving the solubilitycharacteristics of the vegetable proteins. More particularly, thepresent invention relates to a method of enzymatically hydrolyzingvegetable proteins, such as raw, natural soy proteins and denatured soyproteins, to reduce the antigenicity of the vegetable proteins whilealso improving the solubility characteristics of the vegetable proteins.

Over the years, researchers have found that soybeans may be processed torecover or extract a number of valuable components, such as soy proteinand soybean oil, from the soy beans. Also, soybeans may be processed toform soy flours high in nutritionally beneficial substances, such asfiber and protein. Such processing of soybeans often include heattreatment for a variety of purposes, such as inactivating destructiveenzymes or inactivating compounds responsible for off-flavors that areunpalatable to humans and/or animals.

Soybean processing techniques that employ heat frequently causedenaturation of proteins present in resulting soy component andproducts. The degree of protein denaturation depends upon the durationof heat and the temperature profile during the heating, among otherfactors. Additionally, some proteins in soybeans are more susceptible todenaturation at particular heating conditions than are other soybeanproteins. Nonetheless, denaturation of soy proteins is problematic sincedenatured proteins typically exhibit greatly diminished solubility inwater and aqueous solutions.

Many soy products, such as soy flour, soy flakes, and soy meal, areavailable and are commonly used for production of animal feeds and foodproducts for human consumption. However, any such soy products that havebeen heat processed to a substantial degree have also undergonesubstantial soy protein denaturation and, consequently, frequently havea Protein Dispersability Index (subsequently referred to as “PDI”) onthe order of about 20 or even less. The PDI is a measure of proteinsolubility (and consequently a measure of protein dispersability) inwater. The PDI decreases as the level of protein denaturation in a soycomponent or product increases, absent further processing of thedenatured protein to enhance the solubility of the denatured protein.Though there are vegetable protein products with relative high PDIs of90 or more, and thus high levels of soluble proteins, these products aretypically very expensive and/or often contain high levels of antigenicproteins.

Heat treating of soybeans and soybean components, although beneficialfor deactivating destructive enzymes and compounds that contribute tounpalatable tastes, nevertheless do little, if anything, to reduce theantigenicity of the heat-processed soybean products. The antigenicity ofa particular substance is directly correlated to the concentration ofantigens present in the substance. Glycinin and β-conglycinin, which arecommonly referred to as antigenic proteins, are two proteins in soybeanproducts that cause the majority of the antigenicity typically observedin soybean products. Consequently, glycinin and β-conglycinin, by theirpresence or absence, predominantly control the level of antigenicity ofa particular soybean product.

Heat-treating and heat-processing typically do not sufficiently reducethe concentration of antigenic proteins, such as glycinin andβ-conglycinin, in a particular proteinaceous material. Other soybeanprocessing techniques exist that may or may not incorporate heattreatment steps. For example, some commercial processing plants employorganic solvents, such as hexane, to extract oil from soy beans or soyproducts, such as soy flakes. The heat that is applied during the oilextraction process causes some denaturation of protein in the soyproducts. The heat is typically employed during the oil extractionprocess for purposes of evaporating the organic solvent. This heatingfor solvent evaporation purposes may cause some reduction of theantigenic protein concentration, though any such reduction is only aninsignificant reduction. The organic solvent, such as hexane, that isemployed in these processes for oil extraction purposes typically doesnot cause the destruction or removal of antigenic proteins, such asglycinin and β-conglycinin. There are other organic solvents that may beemployed in these processes for purposes other than oil extraction. Someof these other organic solvents may even bring about significantreductions of the concentration of antigenic proteins, such as glycininand β-conglycinin, in a particular proteinaceous material.

The destruction of antigenic protein that provides a reduced level ofantigenicity in soybean products is important, since antigens, such asantigenic proteins, when introduced into a human being or into ananimal, frequently cause production of antibodies that lead todevelopment of allergic reactions that in turn reduce the digestibilityof soybean products or cause other nutritional disturbances. Thus, toreduce the opportunity for allergic reactions, it is beneficial toreduce the antigenicity of soybean products by reducing theconcentration of antigenic proteins, such as glycinin and β-conglycinin,in the soybean products.

However, soybean processing techniques that rely on organic solvents,even though beneficial for destruction of antigenic proteins, are not anoptimum solution to the antigenicity issue. First, reducing theantigenicity of soybean products using such solvent-based processingtechniques nevertheless typically leaves the soybean products with highlevels of denatured proteins. These high levels of denatured proteinscontribute to poor protein solubility characteristics in soybeanproducts produced by solvent-based processing techniques. Furthermore,complete removal of the organic solvent from soybean products producedby solvent-based processing techniques is challenging and oftenincomplete, since trace levels of the organic solvent typically remainin the soybean product. Consumers are increasingly aware of researchstudies that raise questions about the effects of trace levels oforganic solvents on human health. Therefore, to raise public perceptionof food quality, it is useful to minimize or even eliminate use oforganic solvents in food processing techniques.

However, other than solvent-based processing techniques, heat-basedprocessing techniques that denature proteins while leaving antigenicproteins intact or substantially intact are the most common soybeanprocessing techniques. Furthermore, other processing techniques, such asgrinding or milling, though not relying upon heating that denaturesproteins, nevertheless, typically leave high and substantial levels ofantigenic protein in the processed soybean components.

Thus, there is a need in the food and animal feed manufacturingindustries for a technique of processing vegetable protein sources, suchas soybeans and soybean components, that reduces the antigenicity insoybean products to reduce the potential for allergic reactions inhumans and animals that consume the soybean products. Furthermore, thereis a need for a food and animal feed processing technique that improvesthe solubility, and thus the dispersability, of denatured proteins invegetable sources of protein, such as soybean products. Enhancedsolubility and dispersability of denatured proteins is necessary toallow production of beverages, such as milk substitutes, milk replacers,and infant formulas, that contain proteins derived from vegetablesources, such as soybeans, and to support production of food productsand animal feeds that incorporate dispersed or emulsified proteinsderived from vegetable sources, such as soybeans. The process of thepresent invention provides an optimum solution to these needs byproviding a product with proteins exhibiting high levels of solubilitywhere the product also contains minimal, if any, levels of antigenicproteins.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a method of treating a proteinaceousmaterial having a first concentration of β-conglycinin. The methodincludes combining the proteinaceous material with an enzyme to form areaction mixture, the reaction mixture initially having a pH of at leastabout 7.0 standard pH units, allowing the enzyme to hydrolyzeβ-conglycinin present in the reaction mixture to form a proteinaceousintermediate, and inactivating the enzyme present in the reactionmixture after a reaction period to form a proteinaceous product. Theproteinaceous product produced by the method has a second concentrationof β-conglycinin that is at least 99 percent less than the firstconcentration of β-conglycinin. The present invention also includes amethod of treating a proteinaceous material, a method of treating aproteinaceous material having a first concentration of glycinin, and amethod of treating a proteinaceous material having a first ProteinDispersability Index.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a size distribution plot of protein fragments with differentmolecular weights present in a vegetable protein source that was used asfeed material in the process of the present invention.

FIG. 2 is a size distribution plot of protein fragments with differentmolecular weights present in a vegetable protein product produced by theprocess of the present invention based upon the feed material of FIG. 1.

FIG. 3 is a plot of pH and viscosity profiles of a pair of differentslurries based on vegetable protein sources during enzymatic hydrolysisof the slurries in accordance with the present invention.

FIG. 4 is a size distribution plot of protein fragments with differentmolecular weights present in another vegetable protein source that wasused as feed material in the process of the present invention.

FIG. 5 is a size distribution plot of protein fragments with differentmolecular weights present in a vegetable protein product produced by theprocess of the present invention based upon the feed material of FIG. 4.

DETAILED DESCRIPTION

The present invention generally relates to a method of reducing theantigenicity of vegetable proteins, while also improving the solubilitycharacteristics of the vegetable proteins. More particularly, thepresent invention relates to a method of enzymatically hydrolyzingvegetable proteins, such as raw, natural soy proteins and denatured soyproteins, to reduce the antigenicity of the vegetable proteins whilealso improving the solubility characteristics of the vegetable proteins.

Briefly, the process of the present invention entails the formation ofan aqueous slurry of one or more vegetable protein materials to form aslurried vegetable protein feed. The slurried vegetable protein feed issubjected to the action of a protease (a “proteolytic enzyme”) toproduce a slurried vegetable protein product. The pH of the slurriedvegetable protein feed and the temperature of the slurried vegetableprotein feed that are selected such that action of the protease onproteins present in the slurried vegetable protein feed is effective (1)to enhance the level of protein solubility in the slurried vegetableprotein product, as compared to the level of protein solubility in theslurried vegetable protein feed, and (2) to reduce the level ofantigenicity in the slurried vegetable protein product, as compared tothe level of antigenicity in the slurried vegetable protein feed.Preferably, the slurried vegetable protein feed, at an alkaline pH, issubjected to the action of an alkaline proteolytic enzyme at atemperature of about 60° C. or less, to produce the slurried vegetableprotein product. The slurried vegetable protein product, afterpreparation, is then heated to inactivate the proteolytic enzyme and isthereafter dried to form a powdered vegetable protein product of thepresent invention.

The process of the present invention may be beneficially employed tohydrolyze proteins from any source, such as vegetable protein materials,animal protein materials, marine protein materials, and any combinationof any of these. Some examples of vegetable protein materials areprotein materials derived from soybeans, such as soy protein isolate,toasted or untoasted soy flour, soy grits, soy flakes, soy meal, soyprotein concentrates, and any combination of any of these. The vegetableprotein material, such as any of the soybean protein sources listedabove, maybe defatted, reduced fat, or full fat vegetable proteinmaterials. Some examples of animal protein materials include egg albuminisolate; powdered egg whites; dairy protein materials, such as wheyprotein isolate, whey protein concentrate, and powdered whey; and anycombination of any of these. As with the vegetable protein materials,any animal protein material(s) may be defatted, reduced fat, or full fatin nature. Some examples of marine protein materials includeprotein-containing materials derived from marine creatures, such asfish. As with the vegetable protein materials and the animal proteinmaterials, any marine protein material(s) may be defatted, reduced fat,or full fat in nature.

Though descriptions of the present invention are primarily made in termsof vegetable protein material, it is to be understood that any otherprotein material, such as animal protein materials and marine proteinmaterials, may be substituted in place of vegetable protein material, inaccordance with the present invention, while still realizing benefits ofthe present invention. Likewise, it is to be understood that anycombination of any protein material, such as any combination ofvegetable protein material, animal protein materials, and/or marineprotein materials, may be processed in accordance with the presentinvention, while still realizing benefits of the present invention.

The slurried vegetable protein feed may be prepared by combining thevegetable protein material with water. While the total solidsconcentration in the slurried vegetable protein feed is not critical tothe present invention, the total solids concentration in the slurriedvegetable protein feed preferably ranges between about 10 weight percentand about 35 weight percent, based upon the total weight of the slurriedvegetable protein feed. Total solids concentrations higher than about 35weight percent are less desirable because such higher concentrationsincrease the viscosity of the slurried vegetable protein feed andconsequently may cause difficulties in preparing, mixing and/or handlingthe slurried vegetable protein feed. Total solids concentrations lowerthan about 10 weight percent in the slurried vegetable protein feed areless preferred, because such lower total solids concentrations wouldincrease the size of equipment needed to accomplish the process of thepresent invention and would ultimately require removal of greateramounts of moisture to produce the powdered vegetable protein product ofthe present invention.

After preparation, the temperature of the slurried vegetable proteinfeed is adjusted to a temperature where action of the protease onproteins present in the slurried vegetable protein feed is effective (1)to enhance the level of protein solubility in the slurried vegetableprotein product, as compared to the level of protein solubility in theslurried vegetable protein feed, and (2) to reduce the level ofantigenicity in the slurried vegetable protein product, as compared tothe level of antigenicity in the slurried vegetable protein feed.Preferably, when the protease is an alkaline proteolytic enzyme, theslurried vegetable protein feed is heated to a temperature of about 60°C., or less, such as to a temperature of about 50° C. to about 60° C.

The slurried vegetable protein feed may be held in a batch reactor, suchas a tank or other vessel that is jacketed for circulation of steam, hotwater, or other heating fluid to attain and maintain the desiredtemperature, such as the preferred temperature of about 60° C., or less.Alternatively, the slurried vegetable protein feed may be circulatedfrom the batch reactor, through a heat exchanger, and back into thebatch reactor to heat the slurried vegetable protein feed. As anotheralternative, the water that is blended with the vegetable proteinmaterial to form the slurried vegetable protein feed maybe heated priorto combination of the vegetable protein material and the water. Thebatch reactor containing the slurried vegetable protein feed should beequipped with an agitator that is capable of maintaining the homogeneityof the slurried vegetable protein feed during preparation, pHadjustment, and enzymatic hydrolysis.

After the slurried vegetable protein feed has been heated to the desiredtemperature, such as the preferred temperature of about 60° C., or less,an alkaline agent or an acidic agent, as appropriate, is added to adjustthe pH of the slurried vegetable protein feed. The pH of the slurriedvegetable protein feed is adjusted to a pH that is within the range ofpHs where action of the protease on proteins present in the slurriedvegetable protein feed is effective (1) to enhance the level of proteinsolubility in the slurried vegetable protein product, as compared to thelevel of protein solubility in the slurried vegetable protein feed, and(2) to reduce the level of antigenicity in the slurried vegetableprotein product, as compared to the level of antigenicity in theslurried vegetable protein feed.

After the slurried vegetable protein feed has been heated to the desiredtemperature, such as the preferred temperature of about 60° C., or less,the alkaline agent is preferably added to adjust the pH of the slurriedvegetable protein feed to a pH of about 7.0 standard pH units, or more,such as to a pH of about 7.0 standard pH units to about 10.0 standard pHunits, since the activity of one preferred alkaline proteolytic enzymeis improved within this pH range. More preferably, the pH of theslurried vegetable protein feed is adjusted to a pH of about 8.5standard pH units, or more, such as to a pH of above about 8.5 standardpH units to a pH of about 9.5 standard pH units, since the enzymatichydrolysis reaction has been observed to enhance protein solubilityand/or minimize antigenicity levels when the pH of the slurriedvegetable protein feed is adjusted to this more preferred range. Stillmore preferably, the pH of the slurried vegetable protein feed isadjusted to a pH ranging from about 9.0 standard pH units to about 9.5standard pH units, such as at a pH of about 9 standard pH units, sincethe enzymatic hydrolysis reaction has been observed to enhance proteinsolubility and/or minimize antigenicity levels when the pH of theslurried vegetable protein feed is adjusted to this level.

The alkaline agent is preferably an edible, food grade alkaline agent.Some examples of suitable edible, food grade, alkaline agents includesodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesiumhydroxide. Typically, any alkaline agent that is used will be in theform of an aqueous solution of the alkaline agent, such as an alkaline,aqueous solution containing about 10 weight percent of the alkalineagent in water, based upon the total weight of the aqueous solution, tominimize the potential for over-shooting the desired pH of the slurriedvegetable protein feed.

The acidic agent is preferably an edible, food grade acidic agent. Someexamples of suitable edible, food grade, acidic agents includehydrochloric acid and acetic acid. Typically, any acidic agent that isused will be in the form of an aqueous solution of the acidic agent,such as an acidic, aqueous solution containing about 10 weight percentof the acidic agent in water, based upon the total weight of the aqueoussolution, to minimize the potential for over-shooting the desired pH ofthe slurried vegetable protein feed.

After preparation and pH adjustment of the slurried vegetable proteinfeed, the slurried vegetable protein feed is hydrolyzed to cleaveproteins of the slurried vegetable protein feed into protein fragments(peptides) with smaller molecular weights than the proteins of theslurried vegetable protein feed and to reduce the concentration ofantigenic proteins, such as glycinin and β-conglycinin, originallypresent in the slurried vegetable protein feed. The hydrolysis maybeachieved in a single stage enzymatic hydrolysis reaction that employsone or more proteolytic enzymes. Preferably, the one or more proteolyticenzymes are one or more alkaline proteolytic enzymes.

When the preferred alkaline proteolytic enzyme(s) is employed, theenzymatic hydrolysis reaction begins with the slurried vegetable proteinfeed at a pH of about 7.0 standard pH units, such as at a pH of about7.0 standard pH units to about 10.0 standard pH units; more preferablywith the slurried vegetable protein feed at a pH of about 8.5 standardpH units, or more, such as at a pH above about 8.5 standard pH units toabout 9.5 standard pH units; more preferably with the slurried vegetableprotein feed at a pH within the range of from about 9.0 standard pHunits to about 9.5 standard pH units, such as at a pH of about 9standard pH units. After the enzymatic hydrolysis reaction begins, thepH of the slurried vegetable protein feed is thereafter preferablyallowed to freely change, without any subsequent pH adjustment or pHcontrol. Changes in the pH of the slurried vegetable protein feed arethought to be caused by the enzymatic hydrolysis reaction.

As an optional alternative, as the enzymatic hydrolysis reactionprogresses, the pH of the slurried vegetable protein feed may beadjusted or controlled to remain within the pH range or at the pH of theslurried vegetable protein feed that existed upon initiation of theenzymatic hydrolysis reaction. Preferably, however, such adjustment orcontrol of the pH is not done during the enzymatic hydrolysis reactionbecause (1) such pH adjustment or control may require additional laborand/or equipment, (2) such pH adjustment or control does notsignificantly affect (a) the beneficial protein solubility enhancementor (b) the beneficial antigenicity reduction that are achieved by theprocess of the present invention. Furthermore, such pH adjustment orcontrol does not significantly affect the rates at which the beneficialprotein solubility enhancement or the beneficial antigenicity reductionare achieved by the process of the present invention minimization.However, despite not needing to control pH during the enzymatichydrolysis reaction, the temperature of the slurried vegetable proteinfeed within the batch reactor is maintained at the desired reactiontemperature, such as the preferred reaction temperature of about 60° C.,or less, and agitation is maintained to maintain homogeneity of thecontents of the batch reactor during the enzymatic hydrolysis reaction.

Following addition of the proteolytic enzyme, such as the preferredalkaline proteolytic enzyme, the enzymatic hydrolysis reaction isallowed to proceed at the selected temperature, such as the preferredtemperature of about 60° C., or less, for a period of time that iseffective to modify the proteinaceous components of the slurriedvegetable protein feed in accordance with the present invention and tothe desired degree. Though those of ordinary skill in the art willrecognize that this period of time may vary, depending upon theparticular proteolytic enzyme(s) employed, the activity of theproteolytic enzyme(s), the temperature of the slurried vegetable proteinfeed at the onset of, and during, the enzymatic hydrolysis reaction, andother factors, this period of time will, nevertheless, typically rangefrom about 5 minutes to 120 minutes.

Enzymes that are capable of hydrolyzing proteins are commonly referredto as carbonyl hydrolases. In addition to hydrolyzing peptide bonds ofproteins, carbonyl hydrolases, depending upon the conditions, are oftencapable of hydrolyzing peptide bonds of peptides, ester bonds of fattyacids, and ester bonds of triglycerides. As used herein, a proteingenerally consists of at least about ten individual amino acids, whereasa peptide, which is a protein fragment, generally consists of about twoto about nine individual amino acids. There are both naturally-occurringforms of carbonyl hydrolases and recombinant forms of carbonylhydrolases. Some of the more important types of naturally-occurringcarbonyl hydrolases include, for example, lipases, proteases, such assubtilisins and metalloproteases, and peptide hydrolases. Somenon-exhaustive examples of peptide hydrolases include alpha-aminoacylpeptide hydrolase, peptidyl-amino acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxyl proteinase and metalloproteinase. Somenon-exhaustive exemplary classes of proteases may further include thiol,acid, endo, and exo proteases.

A recombinant carbonyl hydrolase is a carbonyl hydrolase that is notnaturally-occurring. A naturally-occurring carbonyl hydrolase is encodedwith a naturally-occurring DNA sequence. In a recombinant carbonylhydrolase, the DNA sequence that would ordinarily encode the carbonylhydrolase has been modified into a mutant, or non-naturally-occurring,DNA sequence. The mutant DNA sequence encodes a substitution, insertion,and/or deletion of one or more amino acids in the amino acid sequencethat would ordinarily be present in the naturally-occurring carbonylhydrolase. Thus, the presence of the mutant DNA sequence, namely anamino acid sequence not found in nature, causes the carbonyl hydrolasethat includes the DNA sequence to be a non-naturally-occurring, orrecombinant, carbonyl hydrolase. The precursor carbonyl hydrolase of anyparticular recombinant carbonyl hydrolase may itself be either anaturally-occurring carbonyl hydrolase or a recombinant carbonylhydrolase. Suitable methods for modifying the amino acid sequence toyield a recombinant carbonyl hydrolase are disclosed in U.S. Pat. Nos.5,185,258; 5,204,015; 5,700,676; 5,763,257; 5,801,038; and 5,955,350 andin PCT Publication Nos. WO 95/10615 and WO 99/20771.

Enzymes that are capable of hydrolyzing proteins may also be known asproteases, and enzymes that are capable of hydrolyzing peptides may alsobe known as peptide hydrolases. Proteases may also be referred to asproteolytic enzymes. Proteases are a form of carbonyl hydrolases that,under suitable conditions, may cleave peptide bonds of proteins, whereaspeptide hydrolases are a form of carbonyl hydrolases that under suitableconditions, may cleave peptide bonds of peptides. Some proteases, undersuitable conditions, may also cleave peptide bonds of peptides, and thusmay also be characterized as peptide hydrolases. Therefore, a peptidehydrolase may also be a protease. On the other hand, a protease is notnecessarily a peptide hydrolase, though some proteases are in factpeptide hydrolases.

Proteases, like carbonyl hydrolases, may be either naturally-occurringproteases with naturally-occurring DNA sequences or recombinantproteases with mutant DNA sequences. Likewise, peptide hydrolases, likecarbonyl hydrolases, may be either naturally-occurring peptidehydrolases with naturally-occurring DNA sequences or recombinanthydrolases with mutant DNA sequences.

Recombinant proteases and recombinant peptide hydrolases may be directlyderived from naturally-occurring proteases and naturally-occurringpeptide hydrolases), respectively (i.e.: when the recombinant proteaseor recombinant peptide hydrolase is a mutant of the naturally-occurringprotease or the naturally-occurring peptide hydrolase, respectively).Also, recombinant proteases and recombinant peptide hydrolases may beindirectly derived from naturally-occurring proteases andnaturally-occurring peptide hydrolases), respectively (where therecombinant protease or recombinant peptide hydrolase is a second orderrelative of the naturally-occurring protease or the naturally-occurringpeptide hydrolase, respectively, i.e.: when a first recombinant proteaseor first recombinant peptide hydrolase is a mutant of a secondrecombinant protease or a second recombinant peptide hydrolase,respectively, and the second recombinant protease or the secondrecombinant peptide hydrolase is a mutant of the naturally-occurringprotease or the naturally-occurring peptide hydrolase, respectively).

Naturally-occurring proteases (and naturally-occurring peptidehydrolases) are available from many sources, including animal,vegetable, and microbial matter. Recombinant proteases and recombinantpeptide hydrolases may be directly or indirectly derived fromnaturally-occurring proteases and naturally-occurring peptidehydrolases, respectively, with any source, such as an animal, vegetable,or microbial source. Naturally-occurring proteases from any source, suchas an animal, vegetable, or microbial source, maybe employed in theprocess of the present invention, and recombinant proteases that aredirectly or indirectly derived from naturally-occurring proteases andnaturally-occurring peptide hydrolases, respectively, from any source,such as an animal, vegetable, or microbial source, may be employed inthe process of the present invention.

Trypsin and chymotrypsin, which are each pancreatic proteases, are somenon-exhaustive examples of suitable naturally-occurring proteases fromanimal matter that may be employed in the process of the presentinvention. Ficin, bromelain, and papain are some non-exhaustive examplesof suitable naturally-occurring proteases from vegetable matter that maybe employed in the process of the present invention. Bacillus spp.,i.e.: Bacillus licheniformis, Bacillus subtilis, Bacillus alkalophilus,Bacillus cereus, Bacillus natto, and Bacillus vulgatus, which are eachbacterial proteases, and Aspergillus spp., Mucor spp., and Rhizopusspp., which are each examples of fungal proteases, are somenon-exhaustive examples of suitable naturally-occurring microbialproteases that may be employed in the process of the present invention.

A serine protease is a protease that includes a catalytic triad of threeparticular amino acids, namely aspartate, histidine, and serine. Likethe more general protease classification, some serine proteases, underappropriate conditions, act as peptide hydrolases that cleave peptidelinkages of peptides and are consequently also properly classified asserine peptide hydrolases. Both naturally-occurring serine proteases andrecombinant serine proteases may be employed in the process of thepresent invention. Preferably, any naturally-occurring serine proteasesand any recombinant serine proteases that are employed in the process ofthe present invention also act as serine peptide hydrolases under theconditions employed in the process of the present invention.

A couple of exemplary serine proteases are subtilisins andchymotrypsins. Subtilisins are microbial proteases, and, morespecifically, have both fungal and bacterial origins. On the other hand,chymotrypsins are pancreatic enzymes with an animal origin. In thesubtilisins, the relative order of the catalytic triad of amino acids(aspartate, histidine, and serine), reading from the amino to carboxyterminus of the triad, is aspartate-histidine-serine. In thechymotrypsins, the relative order of the catalytic triad of amino acids(aspartate, histidine, and serine), reading from the amino to carboxyterminus of the triad, is, however, histidine-aspartate-serine. Thus, asubtilisin is a serine protease that has the catalytic triad of aminoacids arranged in the aspartate-histidine-serine order.Naturally-occurring or recombinant subtilisins may be employed in theprocess of the present invention. Preferably, any naturally-occurring orrecombinant subtilisin that is employed in the process of the presentinvention also acts as a peptide hydrolase under the conditions employedin the process of the present invention.

Bacillus subtilisins are subtilisin proteases with a microbial origin.Like the more general protease classification, some bacillussubtilisins, under appropriate conditions, act as peptide hydrolasesthat cleave peptide linkages of peptides and are consequently alsoproperly characterized as bacillus subtilisin peptide hydrolases. Bothnaturally-occurring bacillus subtilisins and recombinant bacillussubtilisins may be employed in the process of the present invention.Preferably, any naturally-occurring bacillus subtilisins and anyrecombinant bacillus subtilisins that are employed in the process of thepresent invention also act as bacillus subtilisin peptide hydrolasesunder the conditions employed in the process of the present invention.

A series of naturally-occurring bacillus subtilisins is known to beproduced and secreted by various microbial species, such as B.amyloliquefaciens, B. licheniformis, B. subtilis, and B. pumilus, forexample. Though the amino acid sequences of the members of thisnaturally-occurring bacillus subtilisin series are not entirelyhomologous, the subtilisins in this series tend to exhibit the same orsimilar type of proteolytic activity, though stability issues do existfor some members of this series. Also, conditions for satisfactoryactivity levels vary somewhat between some members of this series.Furthermore, it is believed that some members of this series exhibitstrong peptide hydrolase activity, whereas other members of this seriesexhibit little if any peptide hydrolase activity. The exemplary bacillussubtilisins provided above may be divided into two groups: (1) thesubtilisins secreted by B. licheniformis (subtilisin Carlsberg) and B.pumilus, which are generally less stable below a pH of about 9.0 and (2)the subtilisins secreted by B. amyloliquefaciens (subtilisin Novo;subtilisin BPN) and by B. subtilis. Both naturally-occurring subtilisinssecreted by B. licheniformis, B. amyloliquefaciens, and B. subtilis, aswell as, recombinant subtilisins that are directly or indirectly derivedfrom any of these naturally-occurring subtilisins may be employed in theprocess of the present invention.

In one preferred form, a recombinant subtilisin that is obtained throughrecombinant means is employed as the proteolytic enzyme in the processof the present invention. As used herein, the term “recombinantsubtilisin” refers to a subtilisin in which the DNA sequence encodingthe subtilisin is modified to produce a mutant DNA sequence that encodesthe substitution, deletion, and/or insertion of one or more amino acidsin the naturally-occurring subtilisin amino acid sequence that wouldotherwise exist. As one non-exhaustive example, the recombinantsubtilisin may have methionine substituted at amino acid residues 50,124, and 222 in place of phenylalanine, isoleucine, and glutamine,respectively.

Recombinant methods to obtain genes that encode eithernaturally-occurring precursor subtilisins or recombinant precursorsubtilisins are known in the art. The methods generally entailsynthesizing labeled probes with putative sequences that encode regionsof the protease of interest, preparing genomic libraries from organismsexpressing the protease of interest, and screening the libraries for thegene of interest by hybridization to the labeled probes. Positivelyhybridizing clones are then mapped and sequenced.

The identified protease gene is then ligated into a high copy numberplasmid. The high copy number plasmid with the ligated protease gene isthen used to transform a host cell and express the protease of interest.This plasmid replicates in hosts in the sense that the plasmid containsthe well-known elements necessary for plasmid replication: (1) apromoter operably linked to the gene of interest (which may be suppliedas the gene's own homologous promoter if the promoter is recognized,i.e., transcribed by the host), (2) a transcription termination andpolyadenylation region (necessary for stability of the mRNA transcribedby the host from the protease gene in certain eucaryotic host cells)that is exogenous or is supplied by the endogenous terminator region ofthe protease gene, and, desirably, (3) a selection gene, such as anantibiotic resistance gene, that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antibiotic-containing media.High copy number plasmids also contain an origin of replication for thehost that thereby enables large numbers of plasmids to be generated inthe cytoplasm without chromosomal limitations. However, it is within thescope of the present invention to integrate multiple copies of theprotease gene into a host genome. This is facilitated by procaryotic andeucaryotic organisms that are particularly susceptible to homologousrecombination.

The following cassette mutagenesis method may also be used to facilitateconstruction of subtilisin variants (recombinant forms of subtilisin)that may be employed in the process of the present invention, althoughother methods known to those of ordinary skill in the art may also beused. First, the naturally-occurring gene encoding the subtilisin isobtained and sequenced in whole or in part. Then, the sequence isscanned for a point at which mutation (deletion, insertion, and/orsubstitution) of one or more amino acids in the encoded enzyme isdesired. The amino acid sequences flanking this desired mutation pointare evaluated for the presence of restriction sites that supportreplacement of a short segment of the gene with an oligonucleotide poolthat, when expressed, will encode various mutants. Such restrictionsites are preferably unique sites within the protease gene to facilitatereplacement of the gene segment. However, any convenient restrictionsite that is not overly redundant in the protease gene may be used,provided the gene fragments generated by restriction digestion may bereassembled in proper sequence. If restriction sites are not present atlocations within a convenient distance from the desired mutation point(from 10 to 15 nucleotides), suitable restriction sites are generated bysubstituting nucleotides in the gene without causing a change in eitherthe reading frame or the amino acids that are encoded in the finalconstruction.

Mutation of the gene to change the sequence of the gene and conform tothe desired sequence is accomplished by M13 primer extension inaccordance with generally known methods. The task of locating suitableflanking regions and evaluating the needed changes to arrive at twoconvenient restriction site sequences is made routine by the redundancyof the genetic code, a restriction enzyme map of the gene, and the largenumber of different restriction enzymes. Note that if a convenientflanking restriction site is available, the above method need be usedonly in connection with the flanking region that does not contain asite.

The gene may be naturally-occurring gene, a variant of anaturally-occurring gene, or a synthetic gene. A synthetic gene encodinga naturally-occurring or mutant precursor subtilisin may be produced bydetermining the DNA and/or amino acid sequence of a precursorsubtilisin. Multiple, overlapping, synthetic single-stranded DNAfragments are thereafter synthesized, which upon hybridization andligation produce a synthetic DNA encoding the precursor protease. Anexample of a synthetic gene construction is set forth in Example 3 ofU.S. Pat. No. 5,204,015. The entire disclosure of U.S. Pat. No.5,204,015 is therefore incorporated herein by reference.

As one non-exhaustive example, a bacillus subtilisin such as B.amyloliquefaciens subtilisin, which is an alkaline bacterial protease,may be mutated by modifying the DNA encoding the B. amyloliquefacienssubtilisin to encode the substitution of one or more amino acids ofvarious amino acid residues within the mature form of the recombinantsubtilisin product. These mutant subtilisins have at least one propertythat is different when compared to the same property of the precursorsubtilisin. Properties that may be modified fall into severalcategories: oxidative stability, substrate specificity, thermalstability, alkaline stability, catalytic activity, pH activity profile,resistance to proteolytic degradation, K_(m), kcat and K_(m) over kcatratio.

Though extended discussion is provided herein about alkaline proteasesthat may be derived from B. amyloliquefaciens, it is to be understoodthat any other alkaline protease, such as alkaline proteases ofAspergillus sp., Dendryphiella sp., Scolebasidium sp., Candidalipolytica, Yarrowia lipolytica, Aureobasidium pullulans; Streptomycessp., like Strepomyces rectus var. proteolyticus NRRL 3150, Streptomycessp. YSA-130, S. diastaticus SS1, S. corchorusii ST36, S. pactum DSM40530; alkalophilic actinomycetes, such as Nocardiopsis dassonvillei,and Oerskovia xanthineolytica TK-1; Pseudomonas aeruginosa, Pseudomonasmaltophila, or Pseudomonas sp. Strain B45; Xanthomonas maltophil; Vibrioalginolyticus, or Vibrio metschnikovii strain RH530; Kurthia spiroforme;Psiloteredo healdi; Halophiles, such as Halobacterium sp., likeHalobacterium halobium ATCC 43214, or Halomonas sp. ES-10, may beemployed in the process of the present invention to realize benefits ofthe present invention.

The alkaline proteolytic enzyme that is employed in the process of thepresent invention is preferably a bacterial alkaline proteolytic enzyme.More preferably, the bacterial alkaline proteolytic enzyme is derivedfrom a genetically modified strain of bacteria belonging to the speciessubtilis of the genus Bacillus. Still more preferably, the bacterialalkaline proteolytic enzyme belongs to the species amyloliquefaciens ofthe genus Bacillus. Even more preferably, the bacterial alkalineproteolytic enzyme belongs to the species amyloliquefaciens of the genusBacillus that is expressed by a genetically modified strain of bacteriabelonging to the species subtilis of the genus Bacillus. As one suitableexample, the alkaline proteolytic enzyme may be the alkaline proteolyticenzyme present in the MULTIFECT® P-3000 enzyme composition that isavailable from Genencor International, Inc. of Santa Clara, Calif.

The enzyme of the MULTIFECT® P-3000 enzyme composition is a bacterialalkaline proteolytic enzyme that belongs to the speciesamyloliquefaciens of the genus Bacillus and is expressed by agenetically-modified strain of bacteria belonging to the speciessubtilis of the genus Bacillus. The enzyme of the MULTIFECT® P-3000enzyme composition is commonly known as a subtilisin. The MULTIFECT®P-3000 enzyme composition includes the bacterial alkaline proteolyticenzyme, along with a carrier (propylene glycol) that is compatible withthe bacterial alkaline proteolytic enzyme. The MULTIFECT® P-3000 enzymecomposition may be combined with the slurried vegetable protein feed atany concentration that is effective to modify the proteinaceouscomponents of the slurried vegetable protein feed in accordance with thepresent invention. As one non-exhaustive example, the MULTIFECT® P-3000enzyme composition may be combined with the slurried vegetable proteinfeed at a ratio ranging from about ½ pound (about 227 grams) of theMULTIFECT® P-3000 enzyme composition per 100 pounds (45.35 kilograms) ofvegetable protein material to about two pounds (about 907 grams) of theMULTIFECT® P-3000 enzyme composition per 100 pounds (45.35 kilograms) ofvegetable protein material.

Though extended discussion is provided herein about proteases that maybe derived from specific sources, it is to be understood that proteases,generally, such as naturally-occurring proteases from any source(including, for example, an animal, vegetable, or microbial source) maybe employed in the process of the present invention, and recombinantproteases that are directly or indirectly derived fromnaturally-occurring proteases and naturally-occurring peptidehydrolases, respectively, from any source (including, for example, ananimal, vegetable, or microbial source) may be employed in the processof the present invention. Also, naturally-occurring serine proteases andrecombinant serine proteases may be employed in the process of thepresent invention.

Additionally, naturally-occurring or recombinant subtilisins maybeemployed in the process of the present invention.

Furthermore, both naturally-occurring bacillus subtilisins andrecombinant bacillus subtilisins maybe employed in the process of thepresent invention. Likewise, both naturally-occurring subtilisinssecreted by B. licheniformis, B. amyloliquefaciens, and B. subtilis, aswell as, recombinant subtilisins that are directly or indirectly derivedfrom any of these naturally-occurring subtilisins may be employed in theprocess of the present invention.

Preferably, any naturally-occurring proteases and any recombinantproteases that are employed in the process of the present invention, nomatter the source or derivation of the naturally-occurring proteases andany recombinant proteases, also act as peptide hydrolases under theconditions employed in the process of the present invention.

As used herein, proteolytic activity is defined as the rate ofhydrolysis of peptide bonds per milligram of active enzyme. Many wellknown procedures exist for measuring proteolytic activity (K. M. Kalisz,“Microbial Proteinases,” Advances in BiochemicalEngineering/Biotechnology, A. Fiechter ed., 1988). Techniques todetermine such activities are well known in the art and may be used inthe present invention for determining an appropriate concentration ofprotease to be employed in the process of the present invention.

Determining the optimum conditions for operation of a protease areroutine for a worker of ordinary skill in the art. Through routinemethods, it is possible to determine the working pH range, the optimumpH, the working temperature range, the optimum temperature range and thepresence of cofactors and enzyme activators necessary to obtain suitableperformance from the protease for the given task. In general, if acertain set of conditions are necessary for a particular application, itis possible to select a protease which has optimal activity under thoseconditions. Subtilisins are generally active in the alkaline range,i.e., at pHs greater than about 7 standard pH units, and at temperaturesfrom about 10° C. to about 80° C.

The alkaline proteolytic enzyme(s) incorporated in the process of thepresent invention may be characterized as a protease that exhibitsproteolytic activity at alkaline pHs, such as at a pH of about 7standard pH units, or more. The specific level of activity of thealkaline proteolytic enzyme(s) should be effective to modify theproteinaceous components of the slurried vegetable protein feed inaccordance with the present invention. Consequently, the process of thepresent invention is not limited to any particular level of activity ofthe alkaline proteolytic enzyme(s).

Following enzymatic hydrolysis of the slurried vegetable protein feed toform the slurried vegetable protein product, the proteolytic enzyme,such as the preferred alkaline proteolytic enzyme, is deactivated byheating the slurried vegetable protein product to a temperature of atleast about 85° C., or more, for a period of at least about one to abouttwo minutes, or more, preferably for a period of about 5 minutes, ormore, and more preferably for a period of about 5 minutes to about 10minutes. Temperatures at or above about 85° C. are usually sufficient toinactivate the proteolytic enzyme, such as the preferred alkalineproteolytic enzyme.

Beyond the objective of inactivating the proteolytic enzyme, the heatingstep and the manner in which the heating step is performed are notbelieved to be critical to achieving the benefits of the presentinvention. Furthermore, the heating step may be achieved by heating theslurried vegetable protein product in the batch reactor or bycirculating the slurried vegetable protein product through a heatexchanger, a jet cooker, or any similar heating device of the typetypically employed for heating food products in the food manufacturingindustry.

Following inactivation of the proteolytic enzyme, such as the preferredalkaline proteolytic enzyme, the slurried vegetable protein product maybe comminuted to ensure that any fibrous material is broken apart priorto drying the slurried vegetable protein product. Alternatively, thecomminution may be carried out prior to inactivating the proteolyticenzyme. In any event, comminution ensures uniformity of the slurriedvegetable protein product and helps to ensure that uniform dryingoccurs. One example of suitable equipment for achieving adequatecomminution is the COMITROL® Model No. 1700 processor that is availablefrom Urschel Laboratories, Inc of Valparaiso, Ind.

Following proteolytic enzyme inactivation and any comminution, theslurried vegetable protein product is dried. The slurried vegetableprotein product may be dried using any drying technique or equipment,such as a drum dryer, a vibrating bed dryer, or any type of flash dryer.However, the slurried vegetable protein product is preferably flashdried because flash drying creates a uniform powdered product. Spraydrying is the most commonly used flash drying technique, though freezedrying may also be employed. Some examples of suitable spray dryersinclude vertical spray dryers (VRS dryers) and horizontal spray dryers(HRS dryers) that are available from C. E. Rogers Co. of Northville,Mich., and tower spray dryers that are available from Niro Inc. ofColumbia, Md. The slurried vegetable protein product may optionally becooled, such as to a temperature of about 65° C., prior to drying. Thedrying step transforms the slurried vegetable protein product intopowdered vegetable protein product.

The enzymatic hydrolysis that is accomplished in accordance with thepresent invention yields a number of different benefits. For example,the enzymatic hydrolysis dramatically decreases the concentration ofboth glycinin and β-conglycinin, the predominant antigenic proteins, inthe powdered vegetable protein product as compared to the concentrationof these antigenic proteins in the vegetable protein material that isused to form the slurried vegetable protein feed. This reduction ofantigenic protein content in the powdered vegetable protein productgreatly reduces the likelihood that use of the powdered vegetableprotein product in animal feed and human food would lead to thedevelopment of allergies and/or difficulties digesting the powderedvegetable protein product.

As used herein, unless otherwise indicated, the concentration ofglycinin in the vegetable protein material is expressed in terms of theweight of glycinin in the vegetable protein material relative to theweight of crude protein in the vegetable protein material, theconcentration of glycinin in the slurried vegetable protein product isexpressed in terms of the weight of glycinin in the slurried vegetableprotein product relative to the weight of crude protein in the slurriedvegetable protein product, and the concentration of glycinin in thepowdered vegetable protein product is expressed in terms of the weightof glycinin in the powdered vegetable protein product relative to theweight of crude protein in the powdered vegetable protein product. Also,as used herein, unless otherwise indicated, the concentration ofβ-conglycinin in the vegetable protein material is expressed in terms ofthe weight of β-conglycinin in the vegetable protein material, theconcentration of β-conglycinin in the slurried vegetable protein productis expressed in terms of the weight of β-conglycinin in the slurriedvegetable protein product relative to the weight of crude protein in theslurried vegetable protein product, and the concentration ofβ-conglycinin in the powdered vegetable protein product is expressed interms of the weight of β-conglycinin in the powdered vegetable proteinproduct relative to the weight of crude protein in the powderedvegetable protein product.

The particular proteolytic enzyme(s) employed in the enzymatichydrolysis of the present invention, such as the preferred alkalineproteolytic enzyme(s), in combination with the conditions present duringthe enzymatic hydrolysis and the enzyme deactivation step of the presentinvention, should be effective (1) to reduce the concentration ofglycinin by at least about 50 percent, more preferably by at least about70 percent, and most preferably by at least about 85 percent, in thepowdered vegetable protein product as compared to the vegetable proteinmaterial and (2) to reduce the concentration of β-conglycinin by atleast 99 percent, more preferably by about 100 percent, and mostpreferably by 100 percent, in the powdered vegetable protein product ascompared to the concentration of β-conglycinin in the vegetable proteinmaterial. Furthermore, the particular proteolytic enzyme(s), such as thepreferred alkaline proteolytic enzyme(s), and the conditions employedduring the enzymatic hydrolysis and the enzyme deactivation step shouldbe effective to reduce the combined concentration of glycinin andβ-conglycinin by at least about 70 percent, more preferably by at leastabout 80 percent, and most preferably by at least about 92 percent inthe powdered vegetable protein product, as compared to the combinedconcentration of glycinin and β-conglycinin in the vegetable proteinmaterial.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units and (2) the period of enzymatic hydrolysisis about 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, the particularproteolytic enzyme employed in the enzymatic hydrolysis of the presentinvention, in combination with the conditions present during theenzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, is preferably effective (a) to reduce the concentration ofglycinin by at least about 50 percent, more preferably by at least about70 percent, and most preferably by at least about 85 percent, in thepowdered vegetable protein product as compared to the vegetable proteinmaterial and/or (b) to reduce the concentration of β-conglycinin by atleast 99 percent, more preferably by about 100 percent, and mostpreferably by 100 percent, in the powdered vegetable protein product ascompared to the concentration of β-conglycinin in the vegetable proteinmaterial.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units and (2) the period of enzymatic hydrolysisis about 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, the particularproteolytic enzyme employed in the enzymatic hydrolysis of the presentinvention, in combination with the conditions present during theenzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, is preferably effective to reduce the combined concentrationof glycinin and β-conglycinin by at least about 70 percent, morepreferably by at least about 80 percent, and most preferably by at leastabout 92 percent in the powdered vegetable protein product, as comparedto the combined concentration of glycinin and β-conglycinin in thevegetable protein material.

Another benefit of the process of the present invention is the enhancedsolubility of the powdered vegetable protein product in water, ascompared to the solubility of the vegetable protein material in water.Besides reducing the antigenicity of the powdered vegetable proteinproduct, the process of the present invention additionally enhances thewater solubility of proteins present in the powdered vegetable proteinproduct, as compared to the water solubility of the proteins present inthe vegetable protein material. The solubility of protein in aparticular sample may be characterized based upon the Protein DispersionIndex (PDI) of the sample.

When the vegetable protein material has a PDI of about 60 percent, ormore, the process of the present invention is effective to increase thePDI of the powdered vegetable protein product, as compared to the PDI ofthe vegetable protein material, by at least about 20 percent (forexample, changing from a starting PDI of about 62 percent to a PDI of atleast about 82 percent), more preferably by at least about 23 percent(for example, changing from a starting PDI of about 62 percent to a PDIof at least about 85 percent), and most preferably by at least about 26percent (for example, changing from a starting PDI of about 62 percentto a PDI of at least about 88 percent). When the PDI of the vegetableprotein material is less than about 60 percent, the process of thepresent invention is effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, is preferably effective toincrease the PDI of the powdered vegetable protein product to at leastabout 70 percent, and is more preferably effective to increase the PDIof the powdered vegetable protein product to at least about 80 percent.

Preferably, when the PDI of the vegetable protein material is about 40percent, or less, the process of the present invention is effective toincrease the PDI of the powdered vegetable protein product that is basedupon the vegetable protein material to greater than about 60 percent, ismore preferably effective to increase the PDI of the powdered vegetableprotein product to at least about 70 percent, and is still morepreferably effective to increase the PDI of the powdered vegetableprotein product to at least about 80 percent. More preferably, when thePDI of the vegetable protein material is about 20 percent, or less, theprocess of the present invention is effective to increase the PDI of thepowdered vegetable protein product that is based upon the vegetableprotein material to greater than about 60 percent, is still morepreferably effective to increase the PDI of the powdered vegetableprotein product to at least about 70 percent, and is even morepreferably effective to increase the PDI of the powdered vegetableprotein product to at least about 80 percent.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and (3) thevegetable protein material has a PDI of about 60 percent, or more, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product, as compared to the PDI of the vegetableprotein material, by at least about 20 percent, more preferably by atleast about 23 percent, and most preferably by at least about 26percent.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of less than about 60 percent, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of about 40 percent, or less, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of about 20 percent, or less, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

The enhanced solubility of the powdered vegetable protein product inwater, as compared to the solubility of the vegetable protein materialin water, is believed to be due in significant part to the protein (orpeptide) molecular weight reduction that is achieved in the powderedvegetable protein product, as compared to the protein molecular weightof the vegetable protein material. In this regard, when the vegetableprotein material has an average protein molecular weight in the range ofabout 125 kilodaltons to about 440 kilodaltons, the process of thepresent invention is preferably effective to produce the powderedvegetable protein product with an average protein molecular weight ofabout 7500 Daltons or less, more preferably about 5000 Daltons or less,still more preferably about 2500 Daltons or less, even more preferablyabout 2000 Daltons or less, yet more preferably about 1500 Daltons orless, and most preferably about 1250 Daltons or less. As used herein,the term “average protein molecular weight” means the average molecularweight of both proteins and protein fragments (peptides) in the samplebeing considered.

When (1) the slurried vegetable feed has a pH of at least about 7.0standard pH units, preferably at least about 8.5 standard pH units, morepreferably above about 8.5 standard pH units to about 9.5 standard pHunits, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and (3) thevegetable protein material has an average protein molecular weight inthe range of about 125 kilodaltons to about 440 kilodaltons, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to produce the powdered vegetableprotein product with an average protein molecular weight of about 7500Daltons or less, more preferably about 5000 Daltons or less, still morepreferably about 2500 Daltons or less, even more preferably about 2000Daltons or less, yet more preferably about 1500 Daltons or less, andmost preferably about 1250 Daltons or less.

Furthermore, beyond reducing antigenicity levels in the powderedvegetable protein product and increasing protein solubility in thepowdered vegetable protein product, as compared to antigenicity levelsand protein solubility in the vegetable protein material, the process ofthe present invention additionally tends to reduce off-flavors in thepowdered vegetable protein product, as compared to off-flavors presentin the vegetable protein material.

Thus, three major beneficial aspects of the process of the presentinvention include reducing antigenicity levels in the powdered vegetableprotein product, increasing protein solubility in the powdered vegetableprotein product, and reducing off-flavors in the powdered vegetableprotein product, as compared to the levels of these variables in thevegetable protein material. Consequently, after drying, the powderedvegetable protein product may be employed in a wide variety of foodsubstrates, destined for consumption by both animals and humans, toincrease the nutritional value of the food substrates. For example, thepowdered vegetable protein product may be incorporated in milk replacersfor feeding monogastric mammals, such as human babies and young animalswith only one functioning stomach, such as young calves, while enhancingthe solubility and stability of the powdered vegetable protein productin the milk replacer and reducing chances for allergic reaction in themammals fed the milk replacer. Furthermore, the powdered vegetableprotein product may be incorporated in a number of different humanfoods, such as gelatins, beverages, and other foods that would benefitfrom a highly soluble source of protein with low propensity for allergicinducement.

Property Determination & Characterization Techniques

Various analytical techniques are employed herein. An explanation ofthese techniques follows. All values presented in this document forweight percent dry matter for a particular sample are based on the “asis” form of the sample and are therefore on a “wet basis,” unlessotherwise specified herein. All values presented in this document forcertain other parameters in a sample, namely, weight percent organicmatter, weight percent ash, and weight percent crude protein, are basedon the dry matter weight of the sample and are therefore on a “drymatter” or “dry” basis, unless otherwise specified herein. Furthermore,all values presented in this document for weight percent soluble proteinand for concentrations of glycinin and β-conglycinin in a particularsample are based upon the weight of crude protein in the sample, unlessotherwise specified herein.

pH Determinations

Unless otherwise indicated, all pH determinations recited or specifiedherein are based upon use of the Model No. 59003-00 Digital BenchtoppH/mV Meter that is available from Cole-Parmer Instrument Co. of VernonHills, Ill. using the procedure set forth in the instructionsaccompanying the Model No. 59003-00 Digital Benchtop pH/mV Meter. All pHvalues recited herein were determined at or are based upon a sampletemperature of about 25° C.

Dry Matter Weight Determination

The weight percent of dry matter in a particular sample, based upon thetotal weight of the sample, is calculated after first determining themoisture content in the sample. The weight of moisture in a particularsample is determined by analyzing the sample in accordance with Method#930.15 (4.106) of Official Methods of Analysis, Association of OfficialAnalytical Chemists (AOAC) (16^(th) Ed., 1995). The weight percentmoisture in the sample, based upon the total weight of the sample, isthen calculated by dividing the actual weight of moisture in the sampleby the total weight of the sample and then multiplying the result ofthis division by 100%. The weight percent dry matter in the sample isthen determined by subtracting 100% from the weight percent of moisturein the sample. For example, if a particular sample had a moistureconcentration of 22 weight percent, then the dry matter content of thatsample would be 78 weight percent. The weight percent dry matter in theis also known as the weight percent total solids in the sample.

Ash and Organic Matter Determinations

The weight percent ash, dry basis, in a particular sample is determinedafter first determining the weight of ash in the sample. The weight ofash in a particular sample is determined by analyzing the sample inaccordance with Method #942.05 (4.1.10) of Official Methods Of Analysis,Association of Official Analytical Chemist (AOAC) (16^(th) Ed., 1995).The weight percent ash, dry basis, in the sample is then calculated bydividing the actual weight of ash by the weight of dry matter in thesample, that is determined by Method #930.15 as described above, andthen multiplying this result of this division by 100%. The weightpercent organic matter, dry basis, in the sample is then calculated bysubtracting the weight percent ash, dry basis, in the sample from 100%.Thus, if the weight percent ash, dry basis, in a particular sample is 30weight percent, the weight percent organic matter, dry basis, in thesample is consequently 70 weight percent.

Crude Protein Determination

The weight percent crude protein, dry basis, in a particular sample iscalculated after first determining the actual weight of total protein inthe sample. The actual weight of total protein in the sample isdetermined in accordance with Method #991.20 (33.2.11) of OfficialMethods of Analysis, Association of Official Analytical Chemists (AOAC)(16^(th) Ed., 1995). The value determined by the above method yields“total Kjeldahl nitrogen,” which is equivalent to “total protein,” sincethe above method incorporates a factor that accounts for the averageamount of nitrogen in protein. Total Kjeldahl nitrogen and total proteinare sometimes referred to in the dairy industry as “crude protein.”Consequently, the terms “total Kjeldahl nitrogen,” “crude protein,” and“total protein” are used interchangeably herein. Furthermore, thoseskilled in the art will recognize that the term “total Kjeldahlnitrogen” is generally used in the art to mean “crude protein” or “totalprotein” with the understanding that the above-noted nitrogen factor hasbeen applied.

The weight percent crude protein, dry basis, in the sample is calculatedby dividing the actual weight of crude protein (a.k.a. total Kjeldahlnitrogen) by the weight of dry matter in the sample, that is determinedby Method #930.15 as described above, and then multiplying this resultby 100%. The weight percent crude protein in the sample, based on theorganic matter content of the sample, is calculated by dividing theweight percent crude protein, dry basis, of the sample by the weightpercent organic matter, dry basis, in the sample, determined asdescribed above in Ash and Organic Matter Determinations, andmultiplying the result of this division by 100%.

Protein Dispersability Index (PDI) Determination

This method is used to determine the Protein Dispersability Index (PDI)of a particular sample that contains protein. The Protein DispersabilityIndex is a measure of the soluble protein content in a sample, expressedas a percent, by weight, of the crude protein weight in the sample.Consequently, the Protein Dispersability Index is equivalent to theweight percent of soluble protein in a sample, based upon the weight ofcrude protein in the sample. The Protein Dispersability Index (PDI) of aparticular sample that contains protein may be determined in accordancewith Method No. 46-24 (1995), entitled Protein Dispersability Index, ofthe American Association of Cereal Chemists (AACC). The current addressof the American Association of Cereal Chemists is 3340 Pilot Knob Road,St. Paul, Minn. 55121.

Brookfield Viscosity Determination

Unless otherwise indicated, all viscosities recited herein weredetermined using a Brookfield Model No. DV-II+ viscometer that may beobtained from Brookfield Engineering Laboratories of Middleboro, Mass.Any of spindle nos. 4, 5, and/or 6 that are available from BrookfieldEngineering Laboratories for use with the Model No. DV-II+ viscometermay be used when determining the viscosity of a particular sample.Viscosity determinations were conducted in accordance with the OperatingInstructions manual for the Brookfield Model No. DV-II+ viscometer,unless otherwise indicated herein. Unless otherwise indicated herein,viscosity measurements were determined with the sample at a particulartemperature and, consequently, sample temperatures are provided witheach viscosity determination provided herein.

Protein Fragment Size Analysis by HPLC

The molecular weight distribution (or profile) for proteins and peptidesin different samples may be determined using High Pressure LiquidChromatography (“HPLC”). A Waters High Pressure Liquid Chromatographysystem employing a Waters 510 high pressure pump, a Waters 712 WISPautomatic sample injection system, and a Waters 996 Photodiode Arraydetector may be used. The Waters High Pressure Liquid Chromatographysystem employing the specified components maybe obtained from WatersCorporation of Milford, Mass.

Some non-exhaustive examples of samples that maybe analyzed by this HPLCmethod include supernatant samples obtained after centrifuging asolution of the vegetable protein feed or a solution of the powderedvegetable protein product. The solution of the vegetable protein feed orof the powdered vegetable protein product maybe prepared by blendingtogether about 3.2 grams of the vegetable protein feed or of thepowdered vegetable protein product with about 40 milliliters ofdistilled, deionized water to form a slurry. The slurry is placed in a50 milliliter centrifuge tube and then incubated at 30° C. for aboutthree hours with intermittent mixing. After the three hour incubationperiod, the 50 milliliter centrifuge tube containing the slurry isplaced in a centrifuge. After assuring that the centrifuge is balanced,the centrifuge is operated for 10 minutes at a rate of about 2700revolutions per minute. Then, the supernatant layer that forms in the 50milliliter centrifuge tube when centrifuging the slurry is used as thesample in the HPLC procedure.

In the Waters HPLC system, the Waters 996 Photodiode Array detector isset at 206 nanometers. The stationary phase of the chromatographicsystem is a BioSep SEC-S2000 size exclusion column that may be obtainedfrom PHENOMENEX INC. of Torrance, Calif. The mobile phase of thechromatographic system is a solution of 100 mM sodium phosphate with apH of 6.8. The sample flow rate in the system is set at 1.0 ml/minutefor samples of the vegetable protein feed, and the sample flow rate inthe system is set at 1.0 ml/minute for samples of the powdered vegetableprotein product. The data obtained from the HPLC analysis is printed asa graph showing molecular weight distribution (profile) of proteinfragments, expressed in absorption units, as a function of retentiontime. The molecular weights of proteins and peptides in a sample,expressed in Daltons, maybe determined from a standard curve forproteins and peptides of known molecular weight analyzed by theabove-described HPLC procedure to produce a molecular weight profile forthe sample. The distribution of protein molecular weights for theproteins and peptides in the sample may be averaged to determine theaverage protein molecular weight of the sample.

Glycinin and β-conglycinin Determinations

The determination of Glycinin content and β-Conglycinin content in aparticular sample may be conducted in accordance with the followingprocedure, which is based upon an Enzyme-Linked Immunosorbent Assay(subsequently referred to as “ELISA”). The procedure is conducted infour separate steps: Isolation of Native Glycinin and β-Conglycinin,Antibody Preparation, ELISA Assay, and Calculations.

Isolation of Native Glycinin and β-Conglycinin

Native Glycinin and β-Conglycinin are isolated from a raw defattedsoybean flour composition by placing about three grams of the raw (i.e.:not denatured or enzymatically-degraded) defatted soybean flourcomposition into fifteen milliliters (ml) of a 0.15 molar (M) sodiumchloride (NaCl) solution. The mixture of the flour composition and theNaCl solution are held for about 1 hour at 25° C., while maintaining thepH of the mixture at 6.7 with a 1.0 M sodium hydroxide (NaOH) solution,to form a native Glycinin and β-Conglycinin extract. The NaCl and NaOHreagents are available from Sigma Chemical Company of Saint-QuentinFallavier, France.

Next, the native Glycinin and β-Conglycinin extract is clarified bycentrifugation at 1,100×g for 30 minutes at 20° C. A supernatant of theGlycinin and P-Conglycinin extract obtained after centrifugation is thenfurther purified using gel filtration. About 0.5 ml of the supernatantis applied to a Sephacryl L S300-HR column previously equilibrated witha PBS buffer. The supernatant is separated into 1-ml fractions using aPBS buffer elution rate of about 100 ml per hour. The PBS buffer shouldcontain 0.2 grams of potassium chloride (KCl) per liter, 0.2 grams ofpotassium di-hydrogen phosphate (KH₂PO₄) per liter, 8.0 grams of sodiumchloride (NaCl) per liter, 1.14 grams of di-sodium hydrogen phosphate(Na₂HPO₄) per liter, and 0.1 grams of Kathon per liter.

The Sephacryl L S300-HR column is available from Pharmacia of SaintQuentin-en-Yvelines, France, while the various PBS reagents areavailable from Sigma Chemical Company of Saint-Quentin Fallavier,France. Individual purified native Glycinin and β-Conglycinin fractionsare recovered by gel filtration as single peaks at elution volumes thatcorresponded to molecular weights (MW) of 340-440 kiloDaltons (kD) forGlycinin, and 180-230 kD for β-Conglycinin. The purified native Glycininfraction and the purified native β-Conglycinin fraction are stored at−20° C. until required.

The purity of the native Glycinin fraction and the purity of the nativeβ-Conglycinin fraction are confirmed using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Mini-gels (80millimeters (mm)×90 mm) include a 12.5 weight percent acrylamideseparating gel and a 4 weight percent acrylamide stacking gel. Proteinloadings are 5 microgram (μg) of protein per track for the nativeGlycinin fraction and also for the native β-Conglycinin fraction.SDS-PAGE is performed in the presence of a Laemmli buffer system thatincludes Tris-glycine containing 25 millimolar (mM) Tris, 192 mM glycineand, 2 grams of SDS per liter, at a pH of 8.3 under reducing conditionsof about 2 weight percent mercaptoethanol. Molecular weight standardsare also loaded in a separate well. Electrophoresis is performed for 1.5hours at 40 mA. Gels are stained for protein using 0.25 percentCoomassie brilliant blue R250 in methanol:acetic acid:water (5:1:4vol/vol/vol). The SDS-PAGE reagents described above are available fromSigma Chemical Company of La Verpilliere, France.

Antibody Preparation

Antisera are produced in New Zealand White Rabbits that were supplied byRanch Rabbits Ltd of Capthorn, Sussex. The antisera are produced againstthe purified native Glycinin and β-Conglycinin obtained in accordancewith the method described above in the section of this document entitled“Isolation of Glycinin and β-Conglycinin.”

Antisera for Glycinin are produced by emulsifying one (1) milligram (mg)of the purified native Glycinin in 1.0 ml of Freund's complete adjuvant.About 0.7 ml of this Glycinin-based emulsion is administeredintramuscularly to the rabbits on two or three occasions over a five toseven week period. Antisera for β-Conglycinin are produced byemulsifying one (1) milligram (mg) of the purified native β-Conglycininin 1.0 ml of Freund's complete adjuvant. About 0.7 ml of thisβ-Conglycinin-based emulsion is administered intramuscularly to therabbits on two or three occasions over a five to seven week period.

ELISA Assay

Unless otherwise indicated, all reagents used to perform the ELISA Assaymay be obtained from Sigma Chemical Corporation of Saint-QuentinFallavier, France.

1. Sample Extraction

Soybean proteins are extracted for about 1.5 hours from a sample (alsoreferred to herein as the “test protein sample”) of the soybean proteincomposition under consideration using 100 volumes of a borate buffersolution at room temperature of about 22° C. The borate buffer solutionhas a pH of about 8.0 units and contains 100 mM Sodium Perborate(Na₂BO₃) and 0.15 M NaCl. The soybean protein extract obtained from thetest protein sample (also referred to herein as the “test protein sampleextract”) is clarified by centrifugation at 20,000×g for 15 minutes.

2. Glycinin Determination by ELISA Assay

a. Initial Plate Preparation

Two NUNC Immunoplate I microtitration plates, obtained from GibcoEurope, Paisley, United Kingdom, are coated with a solution containingpurified native Glycinin obtained in accordance with the methoddescribed above in the section of this document entitled “Isolation ofGlycinin and β-Conglycinin.” One of the coated plates is used fordetermining the Glycinin content of the test protein sample and one ofthe coated plates is used for determining the Glycinin content of theprotein standards samples.

Prior to coating the two plates, the purified native Glycinin isdissolved in a buffer of 50 mM sodium carbonate buffer at a pH of 9.6 toform a buffered solution containing 1 μg of purified native Glycinin perml of the buffered solution. The two plates are then coated with thepurified native Glycinin by adding 0.3 ml of the buffered solution ineach well of the plates. The two coated plates are then incubated for 16hours at 4° C. After incubation, the two coated plates are washed threetimes with a solution of TWEEN® surfactant and sodium chloride. Afterwashing, the coated and incubated plates are blotted and stored at −20°C. for no longer than 4 weeks.

b. Test Protein Sample

One of the coated and incubated plates prepared in subsection a. aboveentitled “Initial Plate Preparation” is employed in the ELISA assay ofthe Test Protein Sample. The Glycinin antisera obtained in accordancewith the method described above (see section above entitled “AntibodyPreparation”) is diluted to a ratio of about 1:32,000 (v/v) with PBS.Equal volumes of the test protein sample extract (see section 1. aboveentitled “Sample Extraction”) and the diluted antisera are combined toform a mixture. Two hundred (200) μl of the mixture are added to eachwell of the coated plate. The coated plate is then incubated at 37° C.for 4 hours. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has been diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate at the various wellsof the plate is then read at a detection wavelength of 405 nanometersfor 10 seconds using an Argus 300 plate reader from Packard InstrumentsCompany of Meriden, Conn.

c. Protein Standards Samples

One of the coated and incubated plates prepared in subsection a. aboveentitled “Initial Plate Preparation” is employed in the ELISA assay ofthe protein standards samples. The Glycinin antisera obtained inaccordance with the method described above (see section above entitled“Antibody Preparation”) is diluted to a ratio of about 1:32,000 (v/v)with PBS.

A standard solution of Glycinin in PBS at a concentration of 2 mg ofGlycinin per ml of the standard solution is diluted to give a range ofdifferent glycinin standards ranging from 100 nanograms (ng) of glycininper ml to 1 mg of Glycinin per ml. The number of different glycininstandards may, as an example, be equal to the number of wells that areincluded in the plate.

For each of the individual glycinin standards, equal volumes of theparticular glycinin standard and the diluted antisera are combined toform a glycinin standard/antisera mixture. Therefore, the number ofglycinin standard/antisera mixtures is equal to the number of differentglycinin standards. Two hundred (200) μl of each glycininstandard/antisera mixture are added to different wells of the coatedplate. Therefore, as an example, each well of the coated plate maycontain a different one of the glycinin standard/antisera mixtures, ifthe number of different glycinin standards equals the number of wells inthe plate. The coated plate is then incubated at 37° C. for 4 hours.After incubation, the plate is washed three times with an aqueoussolution of NaCl and TWEEN® surfactant.

After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has bee diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate is then read at adetection wavelength of 405 nanometers for 10 seconds using an Argus 300plate reader from Packard Instruments Company of Meriden, Conn.

3. β-Conglycinin Determination by ELISA Assay

a. Initial Plate Preparation

Two NUNC Immunoplate I microtitration plates, obtained from GibcoEurope, Paisley, United Kingdom, are coated with a solution containingpurified native β-Conglycinin obtained in accordance with the methoddescribed above in the section of this document entitled “Isolation ofGlycinin and β-Conglycinin.” One of the coated plates is used fordetermining the β-Conglycinin content of the test protein sample and oneof the coated plates is used for determining the β-Conglycinin contentof the protein standards samples.

Prior to coating the two plates, the purified native β-Conglycinin isdissolved in a buffer of 50 mM sodium carbonate buffer at a pH of 9.6 toform a buffered solution containing 1 μg of purified nativeβ-Conglycinin per ml of the buffered solution. The two plates are thencoated with the purified native β-Conglycinin by adding 0.3 ml of thebuffered solution in each well of the plates. The two coated plates arethen incubated for 16 hours at 4° C. After incubation, the two coatedplates are washed three times with a solution of TWEEN® surfactant andsodium chloride. After washing, the coated and incubated plates areblotted and stored at −20° C. for no longer than 4 weeks.

b. Test Protein Sample

One of the coated and incubated plates prepared in subsection a. aboveentitled “Initial Plate Preparation” is employed in the ELISA assay ofthe Test Protein Sample. The β-Conglycinin antisera obtained inaccordance with the method described above (see section above entitled“Antibody Preparation”) is diluted to a ratio of about 1:16,000 (v/v)with PBS. Equal volumes of the test protein sample extract (seesection 1. above entitled “Sample Extraction”) and the diluted antiseraare combined to form a mixture. Two hundred (200) μl of the mixture areadded to each well of the coated plate. The coated plate is thenincubated at 37° C. for 4 hours. After incubation, the plate is washedthree times with an aqueous solution of NaCl and TWEEN® surfactant.

After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has been diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate at the various wellsof the plate is then read at a detection wavelength of 405 nanometersfor 10 seconds using an Argus 300 plate reader from Packard InstrumentsCompany of Meriden, Conn.

c. Protein Standards Samples

One of the coated and incubated plates prepared in subsection a. aboveentitled “Initial Plate Preparation” is employed in the ELISA assay ofthe protein standards samples. The β-Conglycinin antisera obtained inaccordance with the method described above (see section above entitled“Antibody Preparation”) is diluted to a ratio of about 1:16,000 (v/v)with PBS.

A standard solution of β-Conglycinin in PBS at a concentration of 2 mgof β-Conglycinin per ml of the standard solution is diluted to give arange of different β-Conglycinin standards ranging from 10 nanograms(ng) of β-Conglycinin per ml to 100 μg of β-Conglycinin per ml. Thenumber of different β-Conglycinin standards may, as an example, be equalto the number of wells that are included in the plate.

For each of the individual β-Conglycinin standards, equal volumes of theparticular β-Conglycinin standard and the diluted antisera are combinedto form a β-Conglycinin standard/antisera mixture. Therefore, the numberof β-Conglycinin standard/antisera mixtures is equal to the number ofdifferent β-Conglycinin standards. Two hundred (200) μl of eachβ-Conglycinin standard/antisera mixture are added to different wells ofthe coated plate. Therefore, as an example, each well of the coatedplate may contain a different one of the β-Conglycinin standard/antiseramixtures, if the number of different β-Conglycinin standards equals thenumber of wells in the plate. The coated plate is then incubated at 37°C. for 4 hours. After incubation, the plate is washed three times withan aqueous solution of NaCl and TWEEN® surfactant.

After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has bee diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate is then read at adetection wavelength of 405 nanometers for 10 seconds using an Argus 300plate reader from Packard Instruments Company of Meriden, Conn.

Calculations

A standard curve for glycinin content was prepared based upon the ELISAanalysis of the Glycinin protein standards samples. The axes of thestandard curve included the known glycinin content of the variousGlycinin protein standards samples that were prepared and the opticaldensities measured when using the various Glycinin protein standardssamples. This standard curve for glycinin content was developed usinglinear regression after logit-log transformation. In the standard curve,the glycinin contents of the various glycinin protein standards samplesare stated relative to the crude protein content of the various glycininprotein standards samples, where crude protein contents are determinedusing the methods of Kjedahl (Crude Protein (CP)=[N]×6.25). Theconcentration of glycinin (relative to crude protein content) in testprotein sample(s) are obtained from the standard curve for glycinincontent, based upon the optical densities measured for the test proteinsample(s) when analyzing for glycinin.

Similarly, a standard curve for β-Conglycinin content was prepared basedupon the ELISA analysis of the β-Conglycinin protein standards samples.The axes of the standard curve included the known β-Conglycinin contentof the various β-Conglycinin protein standards samples that wereprepared and the optical densities measured when using the variousβ-Conglycinin protein standards samples. This standard curve forβ-Conglycinin content was developed using linear regression afterlogit-log transformation. In the standard curve, the β-Conglycinincontents of the various β-Conglycinin protein standards samples arestated relative to the crude protein content of the variousβ-Conglycinin protein standards samples, where crude protein contentsare determined using the methods of Kjedahl (Crude Protein(CP)=[N]×6.25). The concentration of β-Conglycinin (relative to crudeprotein content) in test protein sample(s) are obtained from thestandard curve for β-Conglycinin content, based upon the opticaldensities measured for the test protein sample(s) when analyzing forβ-Conglycinin.

Additional Background Information About the ELISA Procedure

Additional background information about determination of Glycinincontent and β-Conglycinin content in a particular sample in accordancewith the Enzyme-Linked Immunosorbent Assay (“ELISA”) procedure that isprovided herein may be obtained from the following publications, whichare each hereby incorporated by reference herein, in their entirety:

-   1. Lallès, J. P., Plumb, G. W., Mills, E. N. C., Morgan, M. R. A,    Tukur, H. M., and Toullec, R., Antigenic Activity of Some Soyabean    Products Used in Veal Calf Feeding: Comparison Between In Vitro    Tests (ELISA Polyclonal vs Monoclonal) And With In Vivo Data, Pages    281-285 in van der Poel, A. F. B., Huisman, J., and Saini, H. S.,    ed., Recent Advances of Research in AntiNutritional Factors in    Legume Seeds, Publ. No. 70 (1993 Wageningen Pers, Wageningen, The    Netherlands);-   2. Lallès, J. P., Tukur, H. M., Dréau, D. and Toullec, R.,    Contribution of INRA to the Study of Antigenicity of Plant Protein    Used in Young Farm Animal Nutrition. In: Van Oort, M. G. and    Tolman, G. H.: Antigenicity of Legume Proteins. TNO Communications.    25 pp (1992);-   3. Tukur, H. M., Lallès, J. P., Mathis, C., Caugant, I., and    Toullec, R., Digestion of Soybean Globulins, Glycinin, α-conglycinin    and β-conglycinin, in the Preruminant and the Ruminant Calf, Can. J.    Anim. Sci., vol. 73, pp. 891-905 (December 1993);-   4. Lallès, J. P., Tukur, H. M., Toullec, R., and Miller, B. G.,    Analytical Criteria for Predicting Apparent Digestibility of Soybean    Protein in Preruminant Calves, J. Dairy Sci., vol 79, pp 475-482    (1996);-   5. Tukur, H. M.; Lalles, J. P.; Plumb, G. W.; Mills, E. N. C.;    Morgan, M. R. A.; and Toullec, R., Investigation of the Relationship    Between in Vitro Elisa Measures of Immunoreactive Soy Globulins and    in Vivo Effects of Soy Products, Journal of Agricultural and Food    Chemistry, 44 (8) pp. 2155-2161 (1996);-   6. Lalles, J. P., Tukur, H. M., Salgado, P., Mills, E. N. C.,    Morgan, M. R. A., Quillien, L., Levieux, D., and Toullec, R.,    Immunochemical Studies on Gastric and Intestinal Digestion of    Soybean Glycinin and Beta-conglycinin in Vivo, Journal of    Agricultural and Food Chemistry, 47 (7) pp. 2797-2806 (July, 1999);-   7. Lalles, J. P.; Tukur, H. M.; and Toullec, R., Immunochemical    Tests for Measuring Glycinin and Beta-conglycinin Concentrations in    Soyabean Products. Predictive Value for Nitrogen Digestibility and    Soyabean Immunogenicity in the Calf, Annales de Zootechnie (Paris),    46 (3), pp 193-205 (1997), CAB Accession Number: 981400459, BIOSIS    NO.: 199799684390;-   8. Lalles J. P.; Tukur H. M.; and Toullec, R., Immunochemical Tests    for the Determination of Glycinin and Beta-conglycinin Levels in    Soya Products for Calf Milk Replacers, EAAP Publication, vol. 81,    pp. 243-244 (1996), BIOSIS NO.: 199699169689;-   9. Tukur, H. M., Pardal, P. B., Formal, M., Toullec, R., Lalles, J.    P., and Guilloteau, P. Digestibility, Blood Levels of Nutrients and    Skin Responses of Calves Fed Soyabean and Lupin Proteins,    Reproduction Nutrition Development vol. 35 (1) pp. 27-44 (1995);-   10. Toullec, R.; Lalles, J. P.; and Tukur, H. M., Biochemical    Characteristics and Apparent Digestibility of Nitrogen in Soyabeans    in Pre-ruminant Calves (Original Title: Caracteristiques    Biochimiques et Digestibilite Apparente Des Matieres Azotees De Soja    Chez Le Veau Preruminant), ISBN: 2-84148-004-6, pp.229-232, (1994    Institut de l'Elevage, Paris, France);-   11. Lalles, J. P., Tukur, H. M., and Toullec, R., Assessment of the    Antigenicity of Soya Products for Calf Milk Replacers: Which    Immunochemical Tests to Use? (Evaluation De L'antigenicite Des    Produits du Soja Destines Aux Aliments D'allaitement Pour Veaux:    Quels Tests Immunochimiques Utiliser?), p. 135 in Proceedings of the    2nd meeting “Rencontres Autour Des Recherches Sur Les Ruminants” of    the Institut National de la Recherche Agronomique, held in Paris    (France), on Dec. 13 and 14, 1995, (December, 1995, Institut de    L'Elevage, Paris, France), ISBN: 2-84148-016-X;-   12. Toullec, R., Lalles, J. P., and Tukur, H. M., Relationships    Between Some Characteristics of Soybean Products and Nitrogen    Apparent Digestibility in Preruminant Calves (Caracteristiques    Biochimiques et Digestibilite Apparente Des Matieres Azotees De Soja    Chez Le Veau Preruminant), pp. 229-232 of the Proceedings of the    first meeting “Rencontres autour des recherches sur les ruminants”.    of the Institut National de la Recherche Agronomique, held in Paris    (France), on Dec. 1 and 2, 1994, (December, 1994, Institut de    l'Elevage, Paris, France), ISBN: 2-84148-004-6;-   13. Lalles, J. P. and Toullec, R., Soybean Products in Milk    Replacers for Farm Animals: Processing, Digestion and Adverse    Reactions, CAB Accession Number: 991411987;-   14. Lalles, J. P., Heppell, L. M. J., Sissons, J. W., and Toullec,    R., Antigenicity of Dietary Protein from Soyabean Meal and Peas in    the Dairy Calf Throughout Weaning, CAB Accession Number: 920451145;-   15. Dreau, D., Larre, C., and Lalles, J. P. Semi-quantitative    Purification and Assessment of Purity of Three Soybean    Proteins—Glycinin, Beta-conglycinin and Alpha-conglycinin—by    Sds-page Electrophoresis, Densitometry and Immunoblotting, Journal    of Food Science and Technology, India, vol. 31 (6), pp. 489-493    (1994), ISSN: 0022-1155;-   16. Heppell, L. M. J., Sissons, J. W., and Pedersen, H. E., A    Comparison of the Antigenicity of Soybean-based Infant Formulas,    British Journal of Nutrition, vol. 58 (3), pp.393-404 (1987);-   17. Sissons, J. W. and Thurston, S. M., Survival of Dietary Antigens    in the Digestive Tract of Calves Intolerant to Soyabean Products,    Research in Veterinary Science vol. 37 (2): pp. 242-246 (1984);-   18. Sissons, J. W., Nyrup, A., Kilshaw, P. J.; and Smith, R. H.,    Ethanol Denaturation of Soybean Protein Antigens, Journal of the    Science of Food and Agriculture, vol. 33 (8): pp. 706-710 (1982);-   19. Kilshaw, P. J., and Sissons, J. W., Gastrointestinal Allergy to    Soyabean Protein in Preruminant Calves. Allergenic Constituents of    Soyabean Products, Research in Veterinary Science, vol. 27 (3): pp.    366-371 (1979);-   20. Heppel, L. M. J., Determination of milk protein Denaturation by    an Enzyme-Linked Immunosorbent Assay, Pages 115-123 in Morris, B. A.    and Clifford, M. N., eds., Immunoassays in Food Analysis (1985    Elsevier Applied Science publishers, London, England); and-   21. Bush, R. S., Toellec, R., Caugant, I., and Guilloteau, P.,    Effects of Raw Pea Flour on Nutrient Digestibility and Immune    Responses in the Preruminant Calf, J. Dairy Sci., vol. 75, pp.    3539-3552 (1992).-   22. Perez, M. D., Mills, E N Clare, Lambert, N., Johnson, I. T., and    Morgan, M. R. A., The Use of Anti-Soya Globulin Antsera in    Investigating Soya Digestion In Vivo, J. of the Science of Food and    Agriculture, vol. 80, pp. 513-521 (2000).

EXAMPLES

The present invention is more particularly described in the followingexamples which are intended as illustrations only since numerousmodifications and variations within the scope of the present inventionwill be apparent to those skilled in the art.

Example 1

This example demonstrates the effectiveness of the process of thepresent invention for substantially enhancing the Protein DispersabilityIndex (PDI) of soy flakes and for substantially decreasing the contentof antigenic proteins, such as glycinin and β-conglycinin, in soy flakesthat are treated in accordance with the process of the presentinvention. In Example 1, 110 gallons (416.4 liters) {968 pounds (439.1kilograms)} of warm water were added to a 240 gallon (908 liter) tank(subsequently referred to as a “batch reactor”). The batch reactor wasequipped with an agitator. The batch reactor was also equipped with ajacket for accommodating steam or hot water circulation to maintain orchange the temperature of the contents of the batch reactor. With thewater at a temperature of about 50° C., 300 pounds (136.1 kilograms) ofsoy flakes were added to the warm water in the batch reactor under slowagitation to form a homogenous slurry of the soy flakes and water. Thesoy flakes were obtained from Harvest States Oilseed Processing &Refining of Mankato, Minn. After addition of the soy flakes wascompleted, hot water was circulated through the jacket of the batchreactor to raise the temperature of the slurry to about 53° C.

After formation of the slurry of soy flakes and water, the initial pH ofthe slurry was about 6.35 standard pH units. About 12 liters of asolution of 10 weight percent NaOH, based upon the total weight of thesodium hydroxide solution, was added to the slurry to adjust the pH ofthe slurry to about 9.00 standard pH units. The amount of sodiumhydroxide solution that was added boosted the pH of the slurry higherthan desired. Therefore, with the slurry still under agitation, aboutfour liters of an aqueous acid solution containing about 10 weightpercent hydrochloric acid, based upon the total weight of the aqueousacid solution, was gradually added to the agitated slurry until the pHof the slurry was reduced to about 8.48 standard pH units.

About 3 pounds (1360 grams) {about 1500 milliliters} of MULTIFECT®P-3000 enzyme composition were then added to the slurry in the batchreactor while agitating the slurry. Thus, the MULTIFECT® P-3000 enzymecomposition was added to the slurry at a ratio of about one pound (454grams) of MULTIFECT® P-3000 enzyme composition per one hundred pounds(45.35 kilograms) of soy flakes. The MULTIFECT® P-3000 enzymecomposition, which is a dark amber colored liquid, was obtained fromGenencor International, Inc. of Santa Clara, Calif. The addition of theMULTIFECT® P-3000 enzyme composition initiated an enzymatic hydrolysisreaction that was allowed to continue in the batch reactor for a periodof about two hours while maintaining the slurry at a temperature rangingfrom about 53° C. to about 55° C. and while maintaining mild agitationof the slurry.

No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.07standard pH units after the two-hour period of enzymatic hydrolysis. Atthe end of the two hour enzymatic hydrolysis period, steam was passedthrough the jacket of the batch reactor and the slurry was heated toabout 85° C. to inactivate the alkaline proteolytic enzyme. Temperatureand pH details during the two hour period of enzymatic hydrolysis andtemperature details during the heating to inactivate the enzymes areprovided below in Table 1:

TABLE 1 Time Temp Description (minutes) pH (° C.) Start of EnzymaticHydrolysis 0 8.48 53.0 20 55.3 60 7.13 54.6 85 7.08 54.0 Start ofHeating to Inactivate Enzymes 117 7.07 53.2 123 57.6 131 64.4 TargetEnzyme Inactivation Temp. Achieved 150 85.0 Enzyme InactivationCompleted 155 85.0Heating of the slurry to inactivate the alkaline proteolytic enzyme wasbegun at time 117 (minutes). The slurry was held at about 85° C. forabout 5 minutes.

After enzyme inactivation was completed, the slurry was then pumped fromthe batch reactor to a pair of 120 gallon (454 liter) storage tanksequipped with agitators. Cold water was circulated through the jacket ofthe batch reactor during the transfer of the slurry to the storagetanks. Also, en route to the storage tanks, the slurry was passedthrough a COMITROL® Model No. 1700 processor to ensure that any fibrousmaterial in the slurry was broken apart prior to drying. After transferof the slurry through the COMITROL® processor and to the storage tankswas completed, 10 gallons (37.8 liters) of hot tap water was added tothe slurry in one of the storage tanks and 15 gallons (56.8 liters) ofhot tap water was added to the slurry in the other of the storage tankto facilitate subsequent spray drying. After hot water addition wascompleted, the diluted slurry in each of the storage tanks wasintroduced into a vertical spray dryer, supplied by C. E. Rogers Co. ofNorthville, Mich., to produce spray dried soy powder. The recovery ratefor the processing described above in this example was about 90.7%,since 300 pounds (136.1 kilograms) of soy flakes were introduced intothe batch reactor, and 272 pounds (123.4 kilograms) of spray dried soypowder were recovered from the spray dryer.

Samples of the soy flakes that were added as feed to the batch reactorand samples of the spray dried soy powder were analyzed for variousproperties. The result of these properties for the soy flakes and forthe spray dried soy powder are provided in Table 2 below:

TABLE 2 PROPERTY SOY FLAKES SPRAY DRIED Dry matter (weight %) 96.2594.77 Organic matter (weight %, based on dry matter weight) 92.41 89.53Ash (weight %, based on dry matter weight) 7.59 10.47 Crude protein(weight %, based on dry matter weight) 49.41 45.96 CP (weight %, basedon organic matter weight) 53.46 51.33 Soluble protein (weight %, basedon crude protein weight) 66.50 85.10 Immunoreactive glycinin (mg/g CrudeProtein) 469 227.3 Immunoreactive β-conglycinin (mg/g Crude Protein) 3020.046 Glycinin + β-conglycinin (mg/g Crude Protein) 771 227The weight percent of dry matter, organic matter, ash, crude protein,and crude protein in the soy flakes and in the spray dried soy powderwere determined in accordance with the procedures for these variablesset forth above in the Property Determination & CharacterizationTechnique section. The glycinin and β-conglycinin concentrations in thesoy flakes and in the spray dried soy powder were determined inaccordance with the Glycinin and β-conglycinin Determinations techniquethat is described above in the Property Determination & CharacterizationTechniques section.

The results shown in Table 2 demonstrate that, even though the soyflakes used as feed in this example contained little, if any, denaturedprotein, enzymatic hydrolysis in accordance with the present inventionwas nonetheless effective to increase the concentration of solubleprotein by about 28 percent in the spray dried soy powder, as comparedto the concentration of soluble protein in the soybean flakes used asfeed. Also, the enzymatic hydrolysis procedure decreased the glycininconcentration by about 51.5 percent in the spray dried soy powder, ascompared to the glycinin concentration in the soy flakes used as feed.Additionally, the enzymatic hydrolysis reduced the concentration ofβ-conglycinin by about 99.9 percent in the spray dried soy powder, ascompared to the concentration of β-conglycinin in the soy flakes used asfeed. Consequently, the enzymatic hydrolysis was effective to reduce theconcentrations of the principal antigenic proteins (glycinin plusβ-conglycinin) by about 70.5 percent in the spray dried soy powder, ascompared to the concentrations of the principal antigenic proteins(glycinin plus β-conglycinin) in the soy flakes used as feed.

Additionally, the soy flakes and the spray dried soy powder wereanalyzed by high pressure liquid chromatography (HPLC) to detect anyshift in molecular weight distribution of protein fragments in the spraydried soy powder versus the soy flakes that were used as feed. The highpressure liquid chromatography analysis was conducted in accordance withthe procedure set forth above in the Property Determination &Characterization Techniques section. The HPLC results for the soy flakesare provided in the graph of FIG. 1, and the HPLC results for the spraydried soy powder are provided in the graph of FIG. 2. Details showingthe variables used in determining the peak areas and the peak areavalues of each of the nine peaks shown in the graph of FIG. 1 and forthe eight peaks shown in the graph of FIG. 2 are provided below inTables 3 and 4, respectively.

TABLE 3 Peak Retention Time Area Height % % No. (min) (uV × sec) (uV)Area Height 1 6.893 1998958 54950 9.47 10.00 2 7.577 747017 34379 3.546.25 3 8.493 3503407 80267 16.60 14.60 4 9.393 1717441 43824 8.14 7.97 510.327 710860 14281 3.37 2.60 6 11.727 177366 5774 0.84 1.05 7 12.377514409 14148 2.44 2.57 8 13.293 538726 23503 2.55 4.28 9 14.243 11193027278605 53.04 50.68

TABLE 4 Peak Retention Time Area Height % % No. (min) (uV × sec) (uV)Area Height 1 6.467 304828 12772 1.06 2.62 2 6.883 695246 17243 2.413.53 3 8.967 2113479 37925 7.33 7.77 4 10.367 558318 9941 1.94 2.04 511.483 778394 17172 2.70 3.52 6 14.400 23445185 365929 81.29 74.97 715.600 897585 25413 3.11 5.21 8 17.450 49551 1728 0.17 0.35The graphs of FIGS. 1 and 2 maybe readily interpreted when it isrecognized that protein fragments with larger molecular weights show upearlier during the HPLC scan in peaks with shorter retention times andprotein fragments with smaller molecular weights show up later duringthe HPLC scan in peaks with longer retention times. Thus, in the graphof FIG. 2, as compared to the graph of FIG. 1, there was a shift tolarger peak areas at higher retention time as compared to peak areas atsimilar retention times in the graph of FIG. 1. This demonstrates thatthe spray dried soy powder, as represented in the graph of FIG. 2,contained protein fragments with a smaller molecular weight average andprofile as compared to the soy flakes depicted in the graph of FIG. 1.This correlates well with the substantially enhanced soluble proteinconcentration in the spray dried soy powder, as compared to the solubleprotein concentration in the soy flakes.

Example 2

This example demonstrates the effectiveness of the process of thepresent invention for substantially enhancing the protein dispersabilityindex (PDI) of defatted soy flour with a PDI of about 20 that containeda substantial amount of denatured protein. This example demonstrates theeffectiveness of the process of the present invention for substantiallydecreasing the concentration of antigenic proteins, such as glycinin andβ-conglycinin, in the 20 PDI defatted soy flour.

In this example, the 20 PDI soy flour was HONEYSOY® 20 PDI soy flourthat was obtained from Harvest States Oilseed Processing & Refining ofMankato, Minn. The 20 PDI soy flour was combined with warm tap water(50° C.) in several batches at the rate of about 2 pounds (907 grams) of20 PDI flour per gallon (3.78 liters) of warm tap water. Each batch of20 PDI soy flour was processed in a Model No. LTDW liquefier obtainedfrom Breddo Likwifier of Kansas City, Kans. to liquity and slurry themixture of 20 PDI soy flour and water.

Each batch of liquified soy flour/water slurry was transferred from theliquefier into a 250 gallon (946 liter) batch reactor that was identicalto the 250 gallon (946 liter) batch reactor described in Example 1above. A total of 325 pounds (147.4 kilograms) of the 20 PDI soy flourwas combined with a total of 162.5 gallons (615.1 liters) of water inthe soy flour/water slurry that was placed in the batch reactor. Afteraddition of the 20 PDI soy flour and water to the batch reactor wascompleted, hot water was circulated through the jacket of the batchreactor to raise the temperature of the soy flour/water slurry to about56.7° C. The initial pH of the soy flour/water slurry was about 6.67standard pH units, and the initial Brookfield viscosity of the soyflour/water slurry was about 1240 centipoise at 56.7° C.

About 16 liters of a aqueous solution of 10 weight percent NaOH inwater, based on the total weight of the sodium hydroxide solution, wasadded to the soy flour/water slurry to adjust the pH of the soyflour/water slurry to about 9.05 standard pH units. Then, about 3.3pounds (about 1.5 kilograms) {about 1.65 liters} of the MULTIFECT®P-3000 enzyme composition was added to the soy flour/water slurry in thebatch reactor. Thus, the MULTIFECT® P-3000 enzyme composition was addedat a ratio of about one pound (about 454 grams) of the MULTIFECT® P-3000enzyme composition per one hundred pounds (45.35 kilograms) of 20 PDIsoy flour. After addition of the enzyme solution, the temperature of thesoy flour/water slurry was determined to be about 56.7° C. and theBrookfield viscosity of the soy flour/water slurry was determined to beabout 1900 centipoise at the 56.7° C. slurry temperature.

The enzymatic hydrolysis reaction triggered by addition of theMULTIFECT® P-3000 enzyme composition was allowed to continue in thebatch reactor for a period of about 2 hours while maintaining the soyflour/water slurry at a temperature ranging from about 56.7° C. to about60° C. No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.58standard pH units after the two-hour period of enzymatic hydrolysis. ThepH, viscosity, and temperature of the soy flour/water slurry at varioustimes during the two-hour enzymatic hydrolysis reaction are shown inTable 5 below:

TABLE 5 Time Viscosity Temp Description (minutes) pH (cp) (° C.) Startof Enzymatic Hydrolysis 0 9.05 1900 56.7 5 8 200 57.3 15 7.9 150 58.5 307.77 110 58.6 60 7.47 110 59 90 7.64 120 59.3 End of EnzymaticHydrolysis 120 7.58 80 60Thus, the enzymatic hydrolysis reaction caused the Brookfield viscosityof the slurry to fall from about 1900 centipoise, measured at 56.7° C.,to about 80 centipoise, measured at about 60° C.

After the two hour enzymatic hydrolysis period, steam was sent throughthe jacket of the batch reactor to inactivate the alkaline proteolyticenzymes. As the slurry was being heated, several Brookfield viscositydeterminations were made. At 70° C., the Brookfield viscosity of theviscosity was found to be about 110 centipoise, at 80° C. the Brookfieldviscosity of the slurry was found to be about 280 centipoise, and at 90°C. the Brookfield viscosity of the slurry was found to be about 440centipoise. After reaching 90° C., the slurry was held at thetemperature of about 90° C. to about 95° C. for a period of about 10minutes to complete inactivation of the alkaline proteolytic enzyme.After the 10 minute enzyme inactivation period, the Brookfield viscosityof the slurry was determined to be about 420 centipoise at 90° C. andabout 850 centipoise at room temperature (about 70° F.).

After enzyme inactivation was completed, the slurry was cooled andcomminution in similar fashion to the cooling and comminution describedin Example 1 and was thereafter spray dried using a vertical spray dryerobtained from C. E. Rogers Co. to produce spray dried soy flour. Therecovery rate for the processing described above in this example wasabout 93.5%, since 325 pounds (147.42 kilograms) of 20 PDI soy flourwere introduced into the batch reactor, and 304 pounds (137.9 kilograms)of spray dried soy flour were recovered from the spray dryer.

Example 3

This example is similar to Example 2 and consequently demonstrates thecapabilities of the process of the present invention for substantiallyenhancing the Protein Dispersability Index (PDI) of defatted soy flourwith a PDI of about 20 that contains a substantial amount of denaturedprotein and for substantially decreasing the content of antigenicproteins, such as glycinin and β-conglycinin in the 20 PDI defatted soyflour.

HONEYSOY® 20 PDI soy flour was used as the feed material in this exampleas in Example 2. Slurry containing the same ratio of 20 PDI soy flour towater was prepared and liquified as described in Example 2 and placed ina batch reactor that was identical to the batch reactor used in Example2. A total of 175 pounds (79.4 kilograms) of 20 PDI soy flour wascombined with a total of 87.5 gallons (331.3 liters) of water in the soyflour/water slurry that was placed in the batch reactor. After additionof the 20 PDI soy flour and water to the batch reactor was completed,hot water was circulated through the jacket of the batch reactor toraise the temperature of the soy flour/water slurry to about 54° C. Theinitial pH of the slurry in the batch reactor was about 6.56 standard pHunits, and the initial Brookfield viscosity of the slurry was about 2800centipoise at a slurry temperature of about 54° C. About one hour afterpreparation, while still being agitated, the pH of the slurry wasobserved to have dropped to about 6.2 standard pH units.

About 8.5 liters of the 10 weight percent NaOH aqueous solution wasadded to the slurry to adjust the pH of the slurry to about 9.06standard pH units. Then, about 1.6 pounds (725.7 grams) {about 0.8liters} of the MULTIFECT® P-3000 enzyme composition was added to the soyflour/water slurry in the batch reactor. Thus, the MULTIFECT® P-3000enzyme composition was added at a ratio of about one pound (454 grams)of the MULTIFECT® P-3000 enzyme composition per one hundred pounds(45.35 kilograms) of 20 PDI soy flour. After addition of the enzymesolution, the temperature of the soy flour/water slurry was determinedto be about 53.9° C. and the Brookfield viscosity of the soy flour/waterslurry was determined to be about 1730 centipoise at the 53.9° C. slurrytemperature.

The enzymatic hydrolysis reaction triggered by an addition of theMULTIFECT® P-3000 enzyme composition was allowed to continue in thebatch reactor for a period of about 2 hours while maintaining the soyflour/water slurry at a temperature ranging from about 54° C. to about60° C. No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.21standard pH units after the two-hour period of enzymatic hydrolysis. ThepH, viscosity, and temperature of the soy flour/water slurry at varioustimes during the two-hour enzymatic hydrolysis reaction are shown inTable 6 below:

TABLE 6 Time Viscosity Temp Description (minutes) pH (centipoise) (° C.)Start of Enzymatic Hydrolysis 0 9.06 1730 53.9 5 7.58 220 54.8 60 7.3180 58.8 90 7.26 60 59.2 End of Enzymatic Hydrolysis 120 7.21 60 59.2Thus, the enzymatic hydrolysis reaction caused the Brookfield viscosityof the slurry to fall from about 1730 centipoise, measured at 53.9° C.,to about 60 centipoise, measured at about 59.2° C.

After the two-hour enzymatic hydrolysis period, steam was entered intothe jacket of the batch reactor to inactivate the alkaline proteolyticenzymes. After reaching 90° C. the soy flour/water slurry was held atthe temperature of about 90° C. to about 95° C. for a period of about 10minutes to complete inactivation of the alkaline proteolytic enzyme.

Discussion of Results for Examples 2 and 3

Examples 2 and 3 each used the same 20 PDI defatted soy flour as thefeed material upon which enzymatic hydrolysis was conducted. Examples 2and 3 each used the same ratio of 20 PDI defatted soy flour to water inthe slurry that was enzymatically hydrolyzed. Examples 2 and 3 each usedthe same alkaline agent and arrived at approximately the same pH bothbefore and after addition of the alkaline agent and the enzyme solution.Also, Examples 2 and 3 each used the same MULTIFECT® P-3000 enzymecomposition, and the same weight ratio of the MULTIFECT® P-3000 enzymecomposition to 20 PDI soy flour ratio was used in both Examples 2 and 3.A graph that is included as FIG. 3 illustrates how the pH profiles ofthe slurry during the enzymatic hydrolysis reactions track in verysimilar fashion for both Examples 2 and 3. This graph of FIG. 3 alsoillustrates how the viscosity profiles of the slurry during theenzymatic hydrolysis reactions track in very similar fashion for bothExamples 2 and 3. It is noted that the viscosities plotted in FIG. 3were not corrected to a standard temperature, but were insteaddetermined at the temperature of the slurry at the time of sampling.

Samples of the 20 PDI soy flour used as the feed material to behydrolyzed in Examples 2 and 3 above were collected and blended inpreparation for analysis. The blended 20 PDI soy flour sample andsamples of the spray dried soy flour obtained in Examples 2 and 3 wereanalyzed for various properties. The results of these analyses areprovided in Table 7 below:

TABLE 7 EXAMPLE 2 EXAMPLE 3 SPRAY SPRAY SOY DRIED SOY DRIED SOY PROPERTYFLOUR FLOUR FLOUR Dry matter (weight %) 94.0 94.5 93.9 Organic matter(weight %, based on dry matter weight) 93.3 92.1 92.3 Ash (weight %,based on dry matter weight) 6.7 7.9 7.7 Crude protein (weight %, basedon dry matter weight) 48.6 48.0 47.3 CP (weight %, based on organicmatter weight) 52.1 52.1 51.2 Soluble protein (weight %, based on crudeprotein weight) 20.8 78.4 81.5 Immunoreactive glycinin (mg/g CrudeProtein) 71 7.5 21 Immunoreactive β-conglycinin (mg/g Crude Protein) 320 0 Glycinin + β-conglycinin (mg/g Crude Protein) 103 7.5 21The weight percent of dry matter, organic matter, ash, crude protein,and crude protein in the 20 PDI soy flour and in the spray dried soyflour were determined in accordance with the procedures for thesevariables set forth above in the Property Determination &Characterization Technique section. The glycinin and β-conglycininconcentrations in the 20 PDI soy flour and in the spray dried soy flourwere determined in accordance with the Glycinin and β-conglycininDeterminations technique that is described above in the PropertyDetermination & Characterization Techniques section.

The results shown in Table 7 demonstrate that the enzymatic hydrolysisprocedures that were carried out in Examples 2 and 3 were each effectiveto dramatically improve the PDI of the blended 20 PDI soy flour from aPDI of about 20 all the way up to a PDI on the order of about 80 for thespray dried soy flour, specifically, a PDI of 78.4 for Example 2 and aPDI of 81.5 for Example 3. Thus, the enzymatic hydrolysis of Example 2improved the PDI in the spray dried soy flour by about 277 percent, ascompared to the 20 PDI soy flour, whereas the enzymatic hydrolysis ofExample 3 improved the PDI in the spray dried soy flour by about 292percent, as compared to the 20 PDI soy flour. These dramatic increasesin the PDI values for soy flours treated in accordance with the presentinvention graphically illustrate the ability of the present invention toimprove solubilities of vegetable protein matter, such as thosecontaining denatured soy proteins.

Also, the enzymatic hydrolysis procedures of Examples 2 and 3 effecteddramatic decreases in the concentration of one antigenic protein,glycinin. In Example 2, the glycinin concentration in the spray driedsoy flour was about 89 percent less than the glycinin level in the 20PDI soy flour, whereas in Example 3 the decrease in glycininconcentration for the spray dried soy flour was a more modest 70percent, as compared to the glycinin concentration in the 20 PDI soyflour. The inventive enzymatic hydrolysis process was even more dramaticin its effectiveness at treating another antigenic protein, namelyβ-conglycinin. More specifically, in Examples 2 and 3, the enzymatichydrolysis procedure was able to completely eliminate any β-conglycinincontent in the spray dried soy flour produced in Examples 2 and 3, eventhough the 20 PDI soy flour used as feed in these examples had aβ-conglycinin concentration of 32 milligrams per gram of crude protein.Consequently, in Example 2, the enzymatic hydrolysis was effective toreduce the overall concentration of the principal antigenic proteins(glycinin plus β-conglycinin) by nearly 90 percent in the spray driedsoy powder, as compared to the concentration of the principal antigenicproteins in the 20 PDI soy flour. Likewise, the enzymatic hydrolysis waseffective to reduce the concentration of the principal antigenicproteins (glycinin plus β-conglycinin) by about 80 percent in the spraydried soy flour of Example 3, as compared to the concentration of theprincipal antigenic proteins in the 20 PDI soy flour.

Samples of a blend of the 20 PDI soy flour used in Examples 2 and 3 asthe feed material and samples of a blend of the spray dried soy flourproduced in Examples 2 and 3 were analyzed by high pressure liquidchromatography (HPLC) to detect any shift in the spray dried soy flourtoward protein fragments with smaller molecular weights versus the 20PDI soy flour. The high pressure liquid chromatography analysis wasconducted in accordance with the procedure set forth above in theProperty Determination & Characterization Techniques section. The HPLCresults for the blend of 20 PDI soy flour used as feed in Examples 2 and3 are provided in the graph of FIG. 4, and the HPLC results for blend ofspray dried soy flour from Examples 2 and 3 are provided in the graph ofFIG. 5.

The graphs of FIGS. 4 and 5 may be readily interpreted when it isrecognized that protein fragments with larger molecular weights show upearlier during the HPLC scan in peaks with shorter retention times andprotein fragments with smaller molecular weights show up later duringthe HPLC scan in peaks with longer retention times. Thus, in the graphof FIG. 5, as compared to the graph of FIG. 4, there was a shift tolarger peak areas at higher retention times as compared to peak areas atsimilar retention times in the graph of FIG. 4. This demonstrates thatthe blend of spray dried soy flours produced in Examples 2 and 3, asrepresented in the graph of FIG. 5, contained protein fragments with asmaller molecular weight average and profile, as compared to the sampleof blended 20 PDI soy flour feed material from Examples 2 and 3, asdepicted in the graph of FIG. 4. This correlates well with thesubstantially enhanced soluble protein concentration (increased PDI) inthe spray dried soy flour samples of Examples 2 and 3, as compared tothe soluble protein concentration in the 20 PDI soy flours used as feedin Examples 2 and 3.

The graphs of FIGS. 4 and 5, when subject to a regression analysis,further demonstrate the beneficial protein molecular weight reductionachieved by the process of the present invention. Specifically, thisregression analysis revealed that the sample of blended 20 PDI soy flourfeed material from Examples 2 and 3, as depicted in the graph of FIG. 4,includes mostly protein fragments with a molecular weight size rangingfrom about 123 kilodaltons to about 394 kilodaltons. On the other hand,regression analysis revealed that the blend of spray dried soy floursproduced in Examples 2 and 3, as represented in the graph of FIG. 5,includes mostly protein fragments with a molecular weight size belowabout 2400 Daltons, with the actual range extending from about 200Daltons to about 2400 Daltons for the vast majority of the proteinfragments.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. A method of treating a proteinaceous soybean material to be utilizedin an animal feed and having a first combined concentration ofβ-conglycinin and glycinin, the method comprising: combining theproteinaceous soybean material with an alkaline proteinase from Bacillusamyloliquefaciens to form a reaction mixture consisting of a singlestage reaction wherein the single stage reaction period begins when thereaction mixture is formed, the reaction mixture initially having a pHof at least about 7.0 standard pH units and wherein the reaction periodis about five minutes to about two hours; allowing the alkalineproteinase to hydrolyze the glycinin and β-conglycinin present in thereaction mixture at a temperature of about 60° C. or less to form aproteinaceous intermediate; and inactivating the alkaline proteinasepresent in the reaction mixture, defining an end of the single stagereaction period, under conditions sufficient to form a proteinaceousproduct, having a second combined concentration of β-conglycinin andglycinin, which is at least 92 percent less than the first combinedconcentration of β-conglycinin and glycinin.
 2. The method of claim 1wherein the reaction mixture initially has a pH of at least about 8.5standard pH units.
 3. The method of claim 2 wherein the method iseffective to provide a second concentration of β-conglycinin that is atleast 99 percent less than the first concentration of β-conglycinin whenno pH adjustment is made during the reaction period after initiation ofthe hydrolysis.
 4. The method of claim 1 wherein the reaction mixtureinitially has a pH greater than about 8.5 standard pH units.
 5. Themethod of claim 1 wherein the second concentration of β-conglycinin isabout 100 percent less than the first concentration of β-conglycinin. 6.The method of claim 1 wherein the Bacillus amyloliquefaciens is arecombinant subtilisin.
 7. A method of treating a proteinaceous soybeanmaterial to be utilized in an animal feed and having a firstconcentration of glycinin and a first concentration of β-conglycinin,the method comprising: combining the proteinaceous soybean material witha serine proteinase from Bacillus amyloliquefaciens to form a reactionmixture consisting of a single stage reaction wherein the single stagereaction period begins when the reaction mixture is formed, the reactionmixture initially having a pH of at least about 7.0 standard pH unitsand wherein the reaction period is about five minutes to about twohours; allowing the serine proteinase to hydrolyze the glycinin andβ-conglycinin present in the reaction mixture at a temperature of about60° C. or less to form a proteinaceous intermediate; and inactivatingthe serine proteinase present in the reaction mixture, defining an endof the single stage reaction period, under conditions sufficient to forma proteinaceous product, having a second concentration of glycinin andβ-conglycinin, the second concentration of glycinin being greater thanabout 50 percent less than the first concentration of glycinin, and thesecond concentration of β-conglycinin being at least about 99 percentless than the first concentration of β-conglycinin.
 8. The method ofclaim 7 wherein the reaction mixture initially has a pH of at leastabout 8.5 standard pH units.
 9. The method of claim 8 wherein the methodis effective to provide the second concentration of glycinin that is atleast about 50 percent less than the first concentration of glycininwhen no pH adjustment is made during the reaction period afterinitiation of the hydrolysis.
 10. The method of claim 7 wherein thereaction mixture initially has a pH greater than about 8.5 standard pHunits.
 11. The method of claim 7 wherein the second concentration ofglycinin is at least about 70 percent less than the first concentrationof glycinin.
 12. The method of claim 7 wherein the Bacillusamyloliquefaciens is a recombinant subtilisin.
 13. A method of treatinga proteinaceous soybean material to be utilized in an animal feed andhaving a first Protein Dispersability Index, the method comprising:combining the proteinaceous soybean material with an alkaline proteinasefrom Bacillus amyloliquefaciens to form a reaction mixture consisting ofa single stage reaction, wherein the proteinaceous soybean material hasan average protein molecular weight of about 125 kilodaltons to about440 kilodaltons, wherein the single stage reaction period begins whenthe reaction mixture is formed, the reaction mixture initially having apH of at least about 7.0 standard pH units and wherein the reaction isabout five minutes to about two hours; allowing the alkaline proteinaseto hydrolyze protein present in the reaction mixture at a temperature ofabout 60° C. or less to form a proteinaceous intermediate; andinactivating the alkaline proteinase present in the reaction mixture,defining an end of the single stage reaction period, under conditionssufficient to form a proteinaceous product, having a second ProteinDispersability Index, at least about 20 percent greater than the firstProtein Dispersability Index and wherein the proteinaceous product hasan average protein molecular weight of about 7500 daltons or less. 14.The method of claim 13 wherein the first Protein Dispersability Index isat least about 60 percent.
 15. The method of claim 13 wherein the firstProtein Dispersability Index is about 20 percent, or less, and thesecond Protein Dispersability Index is at least about 70 percent. 16.The method of claim 13 wherein the proteinaceous product has an averageprotein molecular weight of about 2500 Daltons or less.
 17. The methodof claim 13 wherein the reaction mixture initially has a pH of at leastabout 8.5 standard pH units.
 18. The method of claim 17 wherein thefirst Protein Dispersability Index is about 20 percent, or less, themethod effective to make the second Protein Dispersability Index atleast about 70 percent when no pH adjustment is made during the reactionperiod after initiation of the hydrolysis.
 19. The method of claim 13wherein the reaction mixture initially has a pH greater than about 8.5standard pH units.
 20. The method of claim 19 wherein the first ProteinDispersability Index is about 20 percent, or less, and the secondProtein Dispersability Index is at least about 70 percent.
 21. Themethod of claim 13 wherein the Bacillus amyloliquefaciens is arecombinant subtilisin.
 22. A method of treating a proteinaceous soybeanmaterial to be utilized in an animal feed and having an initial ProteinDispersibility Index, the method consisting essentially of: combiningthe proteinaceous material with water to form a slurry, theproteinaceous material having a first concentration of glycinin and afirst concentration of β-conglycinin; adjusting the pH of the slurry togreater than about 8.5 standard pH units; combining a serine proteinasewith the slurry, defining the beginning of a single stage reactionperiod; and permitting the serine proteinase to hydrolyze proteincontained in the slurry to form a proteinaceous product at a temperatureof about 60° C. or less until the proteinase is inactivated, defining anend to the single stage reaction, the proteinaceous product having asecond concentration of glycinin greater than about 50 percent less thanthe first concentration of glycinin, and a second concentration ofβ-conglycinin at least about 99 percent less than the firstconcentration of β-conglycinin, the proteinaceous product having asecond Protein Dispersability Index of at least about 20 percent greaterthan the initial Protein Dispersability Index rate.
 23. The method ofclaim 22 wherein the pH of the slurry is adjusted to a pH within therange of about 9.0 standard pH units to about 9.5 standard pH units. 24.The method of claim 22 wherein only a single stage hydrolysis reactionoccurs in the slurry and no pH adjustment is made to the slurry afterthe serine proteinase is combined with the slurry.
 25. A method oftreating a proteinaceous soybean material to be utilized in an animalfeed, the method consisting essentially of: combining the proteinaceoussoybean material having a first concentration of β-conglycinin and afirst concentration of glycinin with an alkaline proteinase fromBacillus amyloliquefaciens to form a reaction mixture consisting of asingle stage reaction wherein the reaction period begins when thereaction mixture is formed, the reaction mixture initially having a pHof at least about 7.0 standard pH units; allowing the proteinase tohydrolyze protein present in the reaction mixture at a temperature ofabout 60° C. or less to form a proteinaceous intermediate; andinactivating the enzyme present in the reaction mixture, defining an endof the reaction period, under conditions sufficient to form aproteinaceous product having a concentration of β-conglycinin that is atleast 99 percent less than the first concentration of β-conglycinin andhaving a concentration of glycinin that is at least 70 percent less thanthe first concentration of glycinin, the proteinase derived from agenetically modified strain of Bacillus amyloliquefaciens.
 26. A methodof treating a proteinaceous soybean material to be incorporated into ananimal feed, the method consisting essentially of: combining theproteinaceous soybean material with an alkaline proteinase from Bacillusamyloliquefaciens to form a reaction mixture consisting of a singlestage reaction, wherein the proteinaceous soybean material has anaverage protein molecular weight of about 125 kilodaltons to about 440kilodaltons, wherein the single stage reaction period begins when thereaction mixture is formed, the reaction mixture initially having a pHof at least about 7.0 standard pH units; allowing the proteinase tohydrolyze protein present in the reaction mixture at a temperature ofabout 60° C. or less to form a proteinaceous intermediate; andinactivating the proteinase present in the reaction mixture, defining anend of the single stage reaction period, under conditions sufficient toform a proteinaceous product, having an average protein molecular weightof about 7500 daltons or less.
 27. The method of claim 26 wherein theproteinaceous material has a first concentration of β-conglycinin andthe proteinaceous product has a second concentration of β-conglycinin,the second concentration of β-conglycinin being at least 99 percent lessthan the first concentration of β-conglycinin.
 28. The method of claim26 wherein the proteinase is expressed by a genetically modified strainof Bacillus amyloliquefaciens.
 29. A method of treating a proteinaceoussoybean material to be utilized as a component of an animal feed andhaving a first combined concentration of β-conglycinin and glycinin, themethod consisting essentially of: combining the proteinaceous soybeanmaterial with a serine proteinase to form a reaction mixture consistingof single stage reaction wherein the single stage reaction period beginswhen the reaction mixture is formed, the reaction mixture initiallyhaving a pH of greater than about 8.5 standard pH units; allowing theproteinase to hydrolyze protein present in the reaction mixture at atemperature of about 60° C. or less to form a proteinaceousintermediate; and inactivating the proteinase present in the reactionmixture, defining an end of the single stage reaction period, underconditions sufficient to form a proteinaceous product, having a secondcombined concentration of β-conglycinin and glycinin being at least 70percent less than the first combined concentration, the proteinase beingproduced from a recombinant derived B. licheniformis, B.amyloliquefaciens, or B. subtilis.
 30. The method of claim 29 whereinthe proteinaceous soybean material has a first concentration ofβ-conglycinin and the proteinaceous product has a second concentrationof β-conglycinin, the second concentration of β-conglycinin being atleast 99 percent less than the first concentration of β-conglycinin. 31.The method of claim 29 wherein the reaction mixture initially has a pHwithin the range of about 9.0 standard pH units to about 9.5 standard pHunits.